To construct the eukaryotic expression vectors of fibroblast growth factor receptor 3(FGFR3)  MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN,and to detect their expressions in human chronic myeloid leukemia(CML) K562 cell line.Methods The full-length FGFR3(fgfr3-WT) and dominant negative FGFR3 (fgfr3-DN)were amplified by polymerase chain reaction (PCR).The two genes were respectively digested with EcoRⅠand BamHⅠ,and then ligated into MSCV/puro to construct the recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN  which were tranduced into K562 cells by LipofectaminTM 2000 after PCR,double digestion and DNA sequencing.The expressions of FGFR3 protein in K562 cells were detected by Western blotting and flow cytometry.
Results The recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN were amplified by PCR method,and the results showed fgfr3-WT of 2 400 bp and fgfr3-DN of 1 200 bp had been successfully cloned into MSCV-puro vector. The 2 400 bp fragment was oblained after double digestion of recombinant plasmid.The sequencing results showed that the size of fgfr3-WT was 2 400 bp which was the same as the sequence from GeneBank.Fgfr3-DN of 1 200 bp was also in conformity with the expected sequence.Compared　 with control  (K562 MSCV) group,the expression level  of FGFR3-WT  in MSCV/puro-fgfr3-WT transfection (K562-WT) group was increased to above 10 times.There was high expression of FGFR3-DN in MSCV/puro-FGFR3-DN  transfection  (K562-DN) group,but there was no expressions in  control(K562 MSCV) group and  K562-WT  group.The flow cytometry results showed that the  high expressions of FGFR3-WT  were     in  57.5%   cells in K562-DN group and  the  high expressions of FGFR3-DN  were  in  41.5%  cells in K562-DN  group.Conclusion The K562 cell lines highly expressing FGFR3-WT and FGFR3-DN are constructed successfully.

To observe the protective effect of Panaxadiol Saponins (PDS) on rabbit heart failure model induced by acute alcohol infusion, and to explore its action mechanism of protecting myocardium.Methods 15 healthy rabbits were randomly divided into control group,model group and PDS group,5 rabbits in each group.The rabbits in control group were given 0.2 g•mL^-1 saline by intravenous drip at constant speed,the rabbits in model group were given 20% ethanol with same method,and the rabbits in PDS group were given 0.025 g•kg^-1　PDS by intravenous injection before intravenous drip of 20% ethanol.The  hemodynamic changes were observed by ventricular intubation； the levels of serum lactate dehydrogenase (LDH),creatine kinase (CK),and creatine kinase isoenzyme (CKMB) were determined by colormetric method.The level of  malondialdehyde (MDA) in myocardial tissue  homogenate,the activities of superoxide dismutase (T-SOD),glutathione peroxidase (GSH-Px) and catalase (CAT)  were also detected. Results Compared with control group,the left ventricular end-diastolic pressure (LVEDP) of the rabbits in model group was significantly decreased at 30 min(P<0.05);the serum LDH,CK and CKMB levels were increased(P<0.05),the MDA level in myocardial tissue homogenate was increased(P<0.05),and the T-SOD,GSH-Px and CAT activities were decreased (P<0.05).Compared with model group,the LVEDP of the rabbits in PDS group was increased,the serum LDH,CK,and CKMB levels were decreased(P<0.05),the MDA level was decreased(P<0.05),and the activities of T-SOD,GSH-Px and CAT were increased(P<0.05).Conclusion PDS has protective effect on heart failure induced by acute alcohol infusion,and its mechanism may be related to the improvement of cardiac peroxidation.

To investigate the cytotoxicity of silica nanoparticles on vascular endothelial cells,and to clarify its action mechanism.Methods The 60 nm silica nanoparticle was selected and the in vitro cultured human umbilical vein endothelial cells (HUVECs) were used as cell model.The HUVECs  were divided into control and silica nanoparticle exposure groups with  concentrations of 12.5,25.0， and 100.00 mg•L^-1.MTT assay was used for the determination of cell viability,lactate dehydrogenase (LDH) release assay for membrane integrity,flow cytometry (FCM) for intracellular reactive oxygen species (ROS) content,and real-time PCR assay for intracellular NF-E2-related factor 2 (Nrf2),heme oxygenase-1 (HO-1),superoxide  dismutase 2 (SOD2) and glutamate-cysteine ligase catalytic subunit (GCLC) mRNA levels.Results The MTT results showed that the cell viabilities in each silica nnaoparticle exposure group were decreased compared with control group in a dose-dependent manner.Upon the silica nanoparticle exposure for 12 h,the cell viability was declined significantly only in 100 mg•L^-1 exposure group compared with control group (P<0.05).When  exposured for 24 h,the cell viabilities in 25.0,50.0, and 100.0 mg•L^-1 exposure groups were declined significantly  compared with control group (P<0.05).Under the exposure to silica nanoparticle with the same dose,the cell viabilities were decreased along with the elongation of exposure time. LDH assay and FCM showed that except for that in 12.5 mg•L^-1 exposure group,both the LDH activities in media and intracellular ROS levels in other exposure groups were increased compared with control group (P<0.05).The results of real-time fluorescence PCR showed that the mRNA levels of Nrf2,HO-1,SOD2 and GCLC in  100 mg•L^-1 silica nanoparticle exposure group were increased significantly compared with control group (P<0.05).Conclusion Silica nanoparticles have toxicity to vascular endothelial cells,which includes reducing cell viability,membrane integrity destruction,induction of ROS generation,and tranSCriptional regulation of redox-related factors.Oxidative damage is one of the mechanisms of vascular endothelial toxicity mediated by silica nanoparticles.
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To investigate the effect of leptin on the secretion of chemokine in THP1 cells and explore its related  mechanism,and to provide basis for research on the role of leptin in immune response.Methods The expressions of Ob-Rb and Ob-Rt in THP1 cells were detected by RT-PCR and flow cytometry (FCM).The THP1 cells at  logarithm growth phase were selected and randomly divided into blank control group and different concentrations(10,50,100,200  μg•L^-1) of leptin groups.Transwell chamber assay was performed to detect the number of invated THP1 cells.The THP1 cells were divided into blank control group and 100  μg•L^-1 leptin group.Western blotting method was carried out to detect the expressions of p-AKT,p-ERK 1/2,and p-STAT3 in THP1 cells.The THP1 cells were divided into blank control group and 100  μg•L^-1 leptin group,100  μg•L^-1 leptin+ DMSO group,100  μg•L^-1 leptin+50  μmol?L-1 AG490 group,100  μg•L^-1 leptin+10  μmol•L^-1 LY294002 group and 100  μg•L^-1 leptin+ 10 mol•L^-1 PD980590 group.RT-PCR and Western blotting methods were performed to detect the expression of IL-8.Results  Ob-Rb and Ob-Rt were highly expressed in THP1 cells.Compared with blank control group,the number of invated THP1 cells was significantly increased in 50,100, and 200  μg•L^-1 leptin groups (P<0.05).Compared with blank control group,the expressions of p-STAT3,p-ERK 1/2 and p-AKT in THP1 cells were up-regulated in 100 ug•L^-1 leptin group(P<0.05).Compared with blank control group,the mRNA and protein expressions of IL8 in THP1 cells  in 50,100,and 200  μg?L-1 leptin groups were remarkably increased(P<0.05);compared with 100  μg•L^-1 leptin group,the expressions of IL-8 in THP1 cells in 100  μg•L^-1 leptin+10 mol•L^-1 PD980590 group and 100  μg•L^-1 leptin+10  μmol•L^-1 LY2 94002 group were decreased(P<0.05),while the expression of IL-8 in 100  μg?L-1 leptin+ 50  μmol•L^-1 AG490 group had no  change(P>0.05).Conclusion leptin can up-regulate the expression of chemokine in THP1 cells,which might be associated with PI3K-AKT and MAPK/ERK 1/2 signaling pathways.

 To explore the influence of tranSCription factor FOXC2 in angiogenesis of breast cancer MCF-7 cells and to clarify the action mechanism of FOXC2 in promoting tumor angiogenesis.Methods FOXC2 gene and empty vector gene were transfected into breast cancer of MCF-7 cell line with FOXC2 lentivirus gene transfection technique to obtain stable transfection cell line.The MCF-7 cells were devided into non-transfected group,empty-vector group and over-expression group.Matrigel assay and Transwell chamber test were used to observe the changes of tube formation and migration ability of human umbilical vein endothelial cells (HUVECs) in MCF-7 cells supernatant in various groups.PT-PCR and Western blotting methods were applied to detect the expressions of FOXC2,DLL4 and Notch1 mRNA and protein.Results Compared with non-tranfected group and empty-vector group,the tube formation and the migration number of HUVECs in FOXC2 over-expression group were increased(P<0.05);the  expressions of FOXC2,DLL4 and Notch1 mRNA and proteins were significantly increased(P<0.05).Conclusion The FOXC2 over-expression in MCF-7 cells can increase the tube formation ability and migration ability of HUVECs,and its mechanism may be related to Notch signaling pathway.

To detect the expression levels of the miR-205 in lung cancer tissue and A549 cells and its targeted gene YES1 using qRT-PCR and  dual fluorescence protein repoter assay system,and to explore the possible mechanism of miR-205 to inhibit the proliferation of lung cancer A549 cells.Methods The expression levels of miR-205 in 10 cases of lung cancer tissue and adjacent normal lung tissue were detected with qRT-PCR.The cell growth curve and colony formation assay were used to determine the proliferation rate of A549 cells after transfected by miR-205 mimics and control mimics.The sequences of YES1 3′UTR (untranslated region) and mutation target sites of YES1 3′UTR were inserted into the plasmid which expressed green fluorescence protein (pcDNA3/EGFP) respectively to construct the green fluorescence protein plasmids of YES1-3′UTR and mut-YES1- 3′UTR.There were six groups in the study: YES1-3′UTR,YES1-3′UTR and miR-205 mimics,YES1-3′UTR and control mimics,mut-YES1-3′UTR,mut-YES1-3′UTR and miR-205 mimics,mut-YES1- 3′UTR and control mimics;after the plasmids expressed red fluorescent protein (pDsRed2 -N1 ) were cotransfected into A549 cells,the extracted protein was detected with  fluorescence spectrophotometer.Results Compared with adjacent normal lung tissue,the expression levels of miR-205 in lung cancer tissue and A549 cells were decreased (P<0.05); the proliferation rate of A549 cells in miR-205 mimics group was lower than that in control mimics group (P<0.05).The fluorescence protein expression level in YES1-3′UTR and miR-205 mimics co-transfected group was lower than that in  YES1-3′UTR and control mimics co-transfected group,the difference was statistically significant (P<0.01). The number of cell colony formation of A549 cells in highly expressed YES1  group  was higher than that in cell control group (P<0.05).Conclusion MiR-205 may inhibit the proliferation of A549 cells  through regulating of the expression of YES1 directly.miR-205 and YES1 are potential therapeutic targets for the biological trea
tment of tumor.

To investigate the protective effect of rhein lysinate (RHL) on the liver of the models with diabetic rats,and to provide basis for research on treatment of fatty liver in the patients 　with  diabetes mellitus.Methods The models of diabetic rats were established by intraperitoneally injecting streptozotocin(STZ).40 rats were divided into control,model,25 mg•kg^-1 RHL,and 50 mg•kg^-1 RHL groups(n=10).The levels of malonaldehyde (MDA) and the
 activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected by  thiobarbituric acid method,pyrogallol autoxidation method,and NADPH coupling method,respectively.The pathological changes of liver tissue were observed by hematoxylin and eosin (HE) staining;the content of fat in liver tissue was observed by Nile red staining;the expression levels of fat synthesis-related proteins were detected by Western blotting method.Results  Compared with control group,the body weight of the rats in model group was decreased and the levels of blood glucose,total cholesterol(TC) and triglyceride(TG) were increased (P<0.05);the activities of SOD and GSH-Px in liver tissue were decreased (P<0.05);there were a plenty of fat vacuoles and fat accumulation in liver tissue.The signal pathway of fat synthesis-related ERK1/2-SREBP-1c was activated in model group;compared with model group,it was inhibited in 25 and 50 mg•kg^-1 RHL groups (P<0.05).Compared with model group,the blood glucose,TC and TG of the rats in 25 and 50 mg•kg^-1RHL groups were decreased (P<0.05);the activities of SOD and GSH-Px were increased (P<0.05);however the body weight had no change.Compared with model group,the fatty vacuoles and the fatty accumulation of liver tissue in 25 and 50 mg•kg^-1 RHL groups were decreased.Conclusion The hepatic protection of RHL is correlated with the inhibition of oxidative stress,fat degeneration and fatty accumulation of liver tissues

To construct an eukaryotic expression vector pcDNA-UCA1a(CUDR) and to observe its expression in bladder cancer UM-UC-2 cells,and to provide experimental basis for study on the relationship between UCA1a(CUDR) gene and bladder cancer.Methods Human total length of UCA1a(CUDR) gene was obtained from the 5′-RACE-Ready cDNA of bladder cancer BLZ-211 cells by PCR and was inserted into pcDNA3.1(＋) vector.pcDNA-UCA1a(CUDR) was identified by digestion with EcoRⅠ and BamHⅠ.The bladder cancer UM-UC-2 cells were transfected stably with the constructed eukaryotic expression vector pcDNA-UCA1a(CUDR).The expressions of UCA1a(CUDR) gene in the UM-UC-2 cells transfected with pcDNA-UCA1a(CUDR) and the UM-UC-2 cells transfected with pcDNA3.1(＋) (control vector) were detected by RT-PCR. Results The inserted fragment with 2 200 bp was successfully amplified,which was in accordance with the expected results.The eukaryotic expression vector pcDNA-UCA1a(CUDR) was constructed successfully after identified by double enzyme digestion and sequencing.The RT-PCR results showed that the expression of UCA1a(CUDR) gene in the cells transfected with pcDNA-UCA1a(CUDR) was significantly increased compared with the cells transfected with pcDNA3.1(＋).Conclusion The eukaryotic expression vector pcDNA-UCA1a(CUDR) is successfully constructed.The UCA1a(CUDR) gene highly expresses in the UM-UC-2 cells transfected with the expression vector.

Abstract:Objective To study the role of glutamate receptor subtypes in nucleus tractus solitarius(NTS) in cardiac-somatic motor reflex (CMR) induced by intrapericardial administration of capsaicin, and to clarify the modulation mechanism of NTS to cardiac nociceptoion.Methods 60 SD rats were randomly divided into  ibotenic (IBO) group,glutamate group,MK-801 group,MCGP group,MK-801+MCPG group and DNQX group.The NTS microinjected with 130 mmol•L^-1  IBO 100 nL, 100，200，500 mmol•L^-1 L-glutamate 100 nL,NMDA receptor antagonist 40 and 60 mmol•L^-1 MK-801 100 nL,metabotropic glutamate receptors antagonist 25 and 50 mmol•L^-1  MCPG 100 nL,25 mmol•L^-1   MCPG  50 nL plus 40 mmol•L^-1  MK-801 50 nL,non-NMDA receptor antagonist 20 and 50 mmol?L-1DNQX 100 nL,respectively.The changes of CMR of the rats in various groups were observed. Results Compared with control group, the CMR of the rats in IBO group  was decreased (P<0.05);the CMR was increased as the concentration increased in glutamate group(P<0.05)；the CMR  in MK-801 and MCPG groups were decreased (P<0.05)；the CMR in MK-801+MCPG  group was decreased (P<0.05);the CMR  in DXQX group had no changes (P>0.05).Conclusion  NTS play an facilictory role in cardiac nociception,and the NMDA receptors and mGluRs receptors mediate this facilitory modulation.
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Abstract:Objective To observe the cardiosomatic motor reflex (CMR) of rats,and to clarify the descending modulation of  nucleus  raphes magnus (NRM) in  cardiac nociception and its pathway.Methods 34 male healthy SD rats were randomly divided into NRM electrical stimulation group (n=8),NRM stimulation combined with  dorsolateral funiculus (DLF) transection group (n=8),NRM stimulation combined with antagonist of 5-hydroxytryptamine (5-HT) methysergide intrathecal administration group (n=18).The rat model of CMR was established through injecting capsaicin into the pericardial sac of the rats in various groups.The electromyogram (EMG) response of dorsal spinotrapezius muscle was used as detection index,and by placing the stimulation electrode in NRM and (or) intrathecal catheterization,the   descending inhibitory modulation of NRM in CMR and the influence of  DLF tansection or methysergide in
trathecal administration in descending inhibitory modulation of NRM were observed.Results Compared with  before stimulation,the EMG response was decreased after NRM electrical stimulation (75 μA) (P<0.05); after bilateral DLF transection,the EMG response after NRM stimulation was significantly increased compared with before DLF transection(P<0.05),and there was no significant difference compared with its control (P>0.05).In addition,after intrathecal administration of methysergide,the EMG response after NRM stimulation was significantly increased compared with before intrathecal treatment of methysergide (P<0.05),but it was still significantly smaller than that of its  control (P<0.05).Conclusion Cardiac nociception evoked by capsaicin stimulation is subject to descending inhibitorymodulation from NRM,and the descending inhibition from NRM is conveyed via the DLF and partially mediated by endogenous 5-HT system.

Abstract:Objective
To investigate the influence of hedysaryum polysaccharide (HPS) in the kidney function and expressions of Glut-1 mRNA and protein in kidney tissue of db/db mice with diabetic nephropathy (DN) and to elucidate its possible action mechanism. Methods 10 db/m mice were taken as normal control group(n=10);50 fueling animal model db/db mice with DN were randomly divided into model group,enalapril group and the low,middle and high doses of HPS groups(n=10).The mice in noral control group and model group were given physioloical saline by gavege;and the mice in the other groups  were respectively given  10 mg•kg^-1•d^-1 enalapril,100,200 and 400 mg•kg^-1•d^-1 HPS by gavage;lasted 8 weeks. Picric acid method was used to determine the serum creatinine(SCr) level of the mice,enzyme coupling rate method was used to determine the blood urea nitrogen (BUN) level,ELISA method was used to determine the urinary microalbumin(UMALB) level, RT-PCR method was performed to detect the expression of Glut-1 mRNA,and Western blotting and mmunohistochemical methods were used to detect the expression of Glut-1 protein. Results Compared with model group,the levels of SCr,BUN,UMALB,the mRNA and protein of Glut-1 expressions were decreased,especially in 400 mg•kg^-1•d^-1 HPS and enalapril groups(P<0.01).The HE and Masson staining results showed that less inflammatory cells infiltration in glomerular of the mice were found,capillary lumens were unobstructed,and the collagen deposition was not obvious in 400 mg•kg^-1•d^-1 group. Conclusion HPS could improve the kidney function of the db/db mice  and inhibit the Glut-1 mRNA and protein expressions obviously,which indicates that HPS could delay the development of DN by inhibiting the Glut-1 expression in the glomerular mesangial cell membrane.
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Abstract:Objective To discuss the morphological changes of neurons infected with street strain of rabies virus(RV), and to explore the mechanism of the neuronal dysfunction caused by RV.Methods In vivo,the C57/BL mice were randomly divided into experimental group(n=10) and control group(n=10).The mice in experimental group were inoculated intracranially at 30 μL 　RV　 suspension (10TCID50),and the mice in control group were inoculated
 with identical volume of cell maintenance medium.Anti-ＲＶ antibody was used to detect the RV antigen distribution in brain tissue of the mice.In vitro,the primary hippocampal neurons were cultured for 1 week and the cells were infected by RV.The proliferation of RV was  detected by immunofluorescence method after infection for 72,96, and 120 h.Results The RV  antigen was found in neurons and neuronal dendrites  of hippocampus CA1  4 d after infection;however,the RV antigen was detected only in neuronal dendrites of hippocampus CA1  7 d after infection.The number of infected neurons was the most 120 h after infection,and the number of infected dendrites was decreased. Conclusion RV can  infect and injure the neuronal   dendrites of  hippocampus CA1 region and lead to neuronal dysfunction of the mice.

Abstract:Objective To construct the engineering bacteria expressing the  recombinant human Kunitz protease inhibitor domain of amyloid protein precursor variant (rhKD/APPvar) in Pichia pastoris, and to establish the methods suitable for large-scale fermentation and purification of rhKD/APPvar. Methods The rhKD/APPvar expression vector was constructed based on the rhKD/APPvar-pPICZα  expression vector.Two restriction  enzyme loci (ApaⅠ and SacⅡ) were added to two flanks of KD/APP and human KD/APP activity center RAM was replaced by the active site of BPTI KAR. After the rhKD/APPvar-pPICZα expression vector was transformed into Pichia pastoris,optimized expression and purification of rhKD/APPvar was performed.The  rhKD/APPvar was purified with  cation exchange chromatography and desalting. Results The results of digestion identification and  DNA sequencing analysis  demonstrated that the recombinant plasmid rhKD/APPvar-pPICZα was successfully  constructed and transfected into pastoris X-33.The SDS-PAGE analysis results indicated that rhKD/APPvar expressed after the induction of methanol and the relative molecular weight was 6 700. After a series of experiments the optimal expression conditions of rhKD/APPvar were obtained as follows: the optimal pH was 6.0 and the optimal induction time point was about the 5th day for the strain. After purified the purity of rhKD/APPvar was about 95%. Conclusion KD/APPvar-pPICZ is successfully constructed; after expression in Pichia pastoris and purification,the  rhKD/APPvar protein is  obtained.

Abstract:Objective To study the effect of metformin on the growth of  megakaryocytic leukemia cell line Dami and to explore the molecular mechanisms of the inhibitory effect of metformin on the proliferation of  Dami.Methods The Dami cells were cultured and divided into control and 1,2,4,8,16 and 32 mmol/L metformin groups.Then MTT test was performed to detect the inhitory rate of  proliferation of  Dami cells after treated with different concentrations of metformin.Flow cytometry was used to examine the distribution of cell cycle,and Western blotting was carried out to analyze  the expressions of Cdc2 and CylinB1 and the phosphorylation of Cdc2.Results The MTT results showed that compared with control group,the inhibitory rates of proliferation of  the  Dami cells in 32 mmol/L metformin groups at  0,24,48,72 and 96 h (35.1%±2.3%,49.7%±5.1%,78.85±0.9%,79.1%±3.0%%, and 85.2%±3.2%) were significantly increased(P<0.01),Furthermore,after metformin treatment for  72 h,the inhibitory rates of proliferation of the Dami cells in  1,2,4,8,16 and 32 mmol/L metformin groups were (33.8±0.3)%,(51.9±0.2)%,(59.4±1.6)%,(65.5±2.0)%,(75.5±0.9)%,and (79.1±3.0)%,respectively. Metformin inhibited the growth of Dami cells in a time- and dose-dependent manner. The flow cytometry results results revealed that compared with control group,the percentages of Dami cells in G2/M phase in 1,2 and 4 mmol/L metformin groups were increased from (26.0±0.5)% to (38.5±1.5)%,(48.4±1.1)%, and (58.2±2.7)%;there was significant difference in the percentages of   Dami cells in G2/M phase between control group and 4 mmol/L metformin group (P<0.01).Western blotting analysis showed that compared with control group,the expressions of Cdc2 and CyclinB were evidently reduced,the phosophorylation of Cdc2 at Tyr15 was up-regulated,and the phosphorylation at Thr161 was down-regulated.Conclusion Metformin can inhibit the growth of Dami cells and induce G2/M arrest,and its mechanism may be related to inhibiting the activation of Cdc2/CyclinB1 complex.

bstract:Objective
 To evaluate the effectiveness of ultrafiltration technology in endotoxin removal from purified recombinant MUC1-MBP fusion protein (MUC1-MBP) and to demonstrate the effect of  ultrafiltration on  endotoxin removal.Methods  CM Sepharose FF weak cation exchange （CM) （CM　group）,CM combined with Phenyl Sepharose 6 FF exchange (C6) (CM+C6  group),CM combined with  ultrafiltration (CM+ultrafiltration  group),and CM combined with C6 and  ultrafiltration (CM+C6+ultrafiltration group) were used to purify the MUC1-MBP from E.coli.and remove endotoxin;the expression level of endotoxin  was detected by Chromogenic End-point Tachypleus Amebocyte Lysate.Results There was a single band at the expected molecular weight of 62 000 by SDS-PAGE analysis.and the purity>96% by Quantity One analysis.The endotoxin levels in CM group and CM +C6 group were quite high and there was no significant difference  between two groups (P>0.05);the endotoxin level in CM+ultrafiltration group was significantly lower than that in CM group,and there was significant difference (P<0.01)；the endotoxin level in  CM+C6+ultrafiltration group was significantly decreased compared with CM+C6 group (P<0.01);there was no significant differences of endotoxin level between CM+ultrafiltration group and CM +C6+ultrafiltration group (P>0.05).Conclusion The effects of CM or CM combined with C6 on endotoxin removal are quite poor,especially C6;CM combined with ultrafiltration are quite effective on endotoxin removal,and   ultrafiltration plays an important role in endotoxin removal.

Abstract:Objective To investigate the effect of 1 - methylhydantoin (1-MH) on asthma and cough animal model,and to clarify its mechanism preliminarily. Methods 50 Wistar rats with ovalbumin-induced asthma were randomly divided into model group,1-MH 20,40,and 80 mg/kg groups,positive control group (aminophylline,60 mg/kg),another 10 Wistar rats served as control group. The levels of interleukin-5 (IL-5), eosinophil chemotactic factor (Eotaxin) and eosinophil (EOS) count in bronchoalveolar lavage fluid (BALF)were measured in various groups. 40 guinea pigs of asthma were randomly divided into model group,1-MH 15,30,and 60 mg/kg groups,positive control group (aminophylline,50 mg/kg);the asthma incubation period of guinea pig was measured in various groups.The bronchus of 10 guinea pigs were selected and put in Krebs solution,and the antispasmodic percentages against histamine phosphate of 1-MH 0.25,0.50,and 1.00 g/L were recorded and calculated. 50 mice of cough were randomly divided into model group,1-MH 25,50,and 100 mg/kg groups,positive control group (codeine,50 mg/kg);the cough incubation period and the  number of cough in the mice were measured in various groups. 40 guinea pigs of cough were randomly divided into model group,1-MH 15，30，and 60 mg/kg groups,positive control group (codeine,20 mg/kg);the cough incubation period and  the number  of cough of  the guinea pigs  in various groups were measured. Results Compared with sensitized model control group,the levels of IL-5, Eotaxin and EOS count in BALF of the rats in 1-MH 40 mg/kg and 80 mg/kg groups were reduced(P<0.05 or P<0.01);compared with acetylcholine-induced asthma model control group,the asthma incubation period of guinea pig in 1-MH 30 mg/kg and 60 mg/kg groups was prolonged(P<0.05 or P<0.01);compared with blank control group,the antispasmodic percentages against histamine phosphate  in 1-MH 0.50 g/L and 1.00 g/L  groups were increased(P<0.01);compared with mouse cough model control group,the cough incubation period of the  mice was prolonged,the cough number of the  mice was decreased in 50 mg?kg-1 and 100 mg?kg-1 groups(P<0.05 or P<0.01);compared with guinea pig cough model control group，the cough incubation period of the  guinea pig was prolonged,the cough number of the guinea pig was decreased in 1-MH 30 mg/kg and 60 mg/kg groups (P<0.05 or P<0.01). Conclusion 1-MH has good antiasthmatic and antitussive effects,which may be related to inhibition of airway inflammation and relaxation of bronchial smooth muscle directly .

Abstract:Objective
To investigate the influence of the eye acupuncture therapy in the expressions of brain derived neurophic factor(BDNF) and tyrosine kinase receptor B(TrkB)  in penumbra brain tissue of ischemic area in the rats with acute cerebral ischemia reperfusion injury, and to explore the related mechanism of protective effect on brain of eye acupunture.Methods 62 SD rats were randomly divided into blank control group (n=11),sham operation group (n=11) and model copy  group (n=40). The rats in copy model group were used to establish models with suture method,and the  successful model    rats (n=32)  were randomly divided into model group（n=16）and eye acupuncture group(n=16).The rats in eye acupuncture group were treated with eye acupuncture intervention;the acupoints were chosen according to human acupoint selection method,the liver,the upper-jiao,the down-jiao district,and renal were selected for acupuncture intervention.After reperfusion for 2 h, the acupuncture was performed once every 8 h,lasted for  10 times;30 min after the last intervention,the rats were sacrificed;the regional ischemia penumbra around the parts of the brain tissue was obtained and the expressions of BDNF and TrkB mRNA and protein  in brain tissue of the rats were detected with RT-PCR and Western blotting method.Results Compared with  blank control group,the expression levels of BDNF and TrkB mRNA and protein  in brain tissue of the rats in sham operation group had no statistical significance（P>0.05）；compared with  sham operation group,the expression levels of BDNF and TrkB mRNA and protein  in brain tissue of the rats in model group were significantly up-regulated(P<0.01);compared with  model group,the expression levels of BDNF and TrkB mRNA and protein  in brain tissue of the rats in eye acupuncture group were significantly up-regulated（P<0.05 or P<0.01）.Conclusion The expressions of BDNF and TrkB are increased after cerebral ischemia and reperfusion injury.Eye acupuncture can protect the brain of MCAO/R rats,which may be related to the up-regulation of the expressions of BDNF and TrkB in the corresponding penumbra.

Abstract:Objective To investigate the influence of prepubertal exposure to estradiol benzoate (EB) in the male reproductive system of the rats and the natural process of tissue repair,and to clarify the possible mechanism of  the reproductive toxicity of exogenous estrogen.Methods Ninety 21-day-old male Wistar rats were randomly  divided into 2 experimental groups (low dose  of EB group and high dose of EB group,n=30) and  control group (n=30).The rats in the experimental groups were injected with  EB dissolved in peanut oil at 15（low dose of EB group） and 15 000 μg/kg（high dose of EB group） respectively,the rats in control group received equal vehicle injection only,once every other day for two weeks from postnatal day(PND)21 to 34.All of them were normally fed after the drug usage was stopped. The testes were harvested at the stages of PND 60 and PND 125(n=15 at each stage). The serum levels testosterone of (T),follicle-stimulating hormone(FSH),luteinizing hormone(LH),prolactin(PRL) and estradiol(E2) of the rats in various groups were detected with radioimmunology method and the weights of the rats in various groups were recorded;the histological changes of the testes tissue were observed with light microscope.Results On PND60,compared with control group,the T levels in low dose of EB group and high dose of EB group were decreased（P<0.05 or P<0.01）;the FSH,LH and E2 levels were increased (P<0.05 or P<0.01),and the PRL levels had no change（P>0.05）;the weights of testes were decreased(P<0.01);the histological  changes of the testes of the rats in experimental groups included seminiferous tubules maldevelopment,decreased cell number of seminiferous epithelia. Compared with low dose of EB group,the T and FSH levels in high dose of EB  group were decreased ( P<0.05 or P<0.01),the E2 and LH levels were increased( P<0.01),the  PRL level had no change（P>0.05）,and the weight of testes was decreased( P<0.01);the diameters of seminiferous tubules were smaller,there was no sperm in high dose  of EB group while there were a few sperms in low dose of EB group.On PND125,compared with control group,the T,FSH and PRL levels in low dose of EB group and high dose of EB group were decreased（P<0.01）,the E2 levels were increased (P<0.01);the LH level in low dose of EB group was increased（P<0.05）,the LH level in high dose of EB group was decreased（P<0.01）,and the weights of testes in high dose of EB group  were decreased(P<0.01); the diameters of seminiferous tubules and the cell number of seminiferous epithelia were increased but not apparent change. Compared with low dose of EB group,the T,LH levels in high dose of EB group were decreased (P<0.01),the E2 and FSH levels were increased(P<0.01),the PRL level had no change（P>0.05）,and the weight of testes was decreased(P<0.01);there was still no sperm in high dose of EB group,the number of sperms was increased in low dose of EB group,but it was  still lower  than that in control group.Conclusion EB is harmful to the reproductive system and can change the normal serum sex hormone levels,even  induces the irreversible injury.

Abstract:Objective To observe the influence of early postoperative feeding in the healing of  intestinal anastomosis in rabbits,and to clarify preliminarily the relationships between early postoperative feeding   after gastrointestinal surgery and gastrointestinal anastomotic fistula formation and healing time in rabbits. Methods 48 rabbits were randomly divided into experimental group and control group,then they were treated with gastrointestinal anastomosis.The rabbits in experimental group were fed with liquid diet 24 h after operation,and the rabbits in control group were fed nothing after operation and supplied by total parenteral nutrition.Two rabbits of each group were selected for exploratory laparotomy on the 3rd,5th,7th,10th and 15th day after operation,and the healing rate of  anastomosis,the anastomotic bursting pressure,the anastomotic breaking strength,and the hychoxyproline level of anastomosis were observed.Results The healing rate of anastomosis in control group was 91.6%(22/24),and the healing rate of anastomosis in experimental group was 95.8%(23/24),there was no significant difference between two groups(P>0.05).The anastomotic bursting pressures of the rabbits in two groups were decreased remarkably at the 72nd hour  after operation,which was the lowest point,and they were increased remarkably on the 5th day after operation,but the anastomic bursting pressure in experimental group was a little lower than that in control group,and it  reached the peak on the 7th day after operation in control group.On the 10th day after operation,the anastomic bursting pressure in control group was a little lower than that on the 7th day after operation,but the anastomic bursting pressure in experimental group reached the peak.There were no significant differences of anastomic bursting pressure at different time points between two groups(P>0.05).The anastomotic breaking strength had no significant difference between two groups at the 72nd hour after operation,both of them reached the lowest points,however the anastomtic breaking strengths in two groups were increased remarkably on the 10th day after operation,and reached the peaks.but there were no significant differences of anastomic breaking strength at different time points between two groups(P>0.05).The hychoxyproline level of anastomosis:in experimental group was a little lower than that in control group at the 72tnd hour after operation,and both of them reached the peaks on the 7th day after operation;but there were no significant differences of hychoxyproline levels of anastomosis at different time points between two groups(P>0.05).Conclusion  Early postoperative feeding can not cause the increase of anasmotic healing time and the incidence rate of gastrointestinal anastomotic fistula.  

Abstract:Objective To investigate the effects of Yindan Xinnaotong Capsule on the osteoponin（OPN）expression in myocardium tissue of the rats after acute myocardial infarction(AMI) and to clarify the mechanism of Yindan Xinnaotong Capsule in alienating rat AMI.Methods 90 Wistar rats were used to establish AMI models by ligating of the left anterior descending coronary artery.The AMI model rats  were  randomly divided into　AMI model group,high dose of Yindan (16 g/kg/d) group,low dose of Yindan(0.08 g/kg/d) group,positive drug control captopril(5 mg/kg/d) group(n=12)；at the same time sham operation group(n=10) was set up,the rats in sham operation group was treated with wearing without ligation.All the rats were administrated for 4 weeks,then the myocardium tissue samples were obtained.The histological changes of myocardium tissue were observed by HE and Masson staining;the DNA fragments of apoptotic cells were detected by TUNEL staining and the apoptotic index(AI)was calculated.The expression of OPN mRNA in non-infarction area was measured by RT-PCR.Results Compared with sham operation group,the AI  of the rats in model group was significantly increased(P<0.05)； compared with model group,the AI of the rats in Yindan groups and captopril group were markedly decreased(P<0.01);compared with captopril group,the AI of the rats in high dose of Yindan group was significantly decreased(P<0.05).Compared with sham operation group,the expression level of OPN mRNA in non-infarction area of the rats in model group was significantly increased(P<0.01);compared with model group,the expression levels of OPN mRNA in non-infarction area of the rats in Yindan groups and captopril group were also decreased significantly(P<0.01);compared with captopril group,the expression level of OPN mRNA non-infarction area of the rats in high dose of Yindan group was significantly decreased(P<0.05).Conclusion Yindan Xinnaotong Capsule could obviously alleviate the apoptosis of myocardium tissue of the rats after acute myocardial infarction and decrease the expression of OPN mRNA in non-infarction area of left ventricle,which indicates that Yindan Xinnaotong Capsule may protect myocardium tissue through decreasing the OPN mRNA  expression.

Abstract:Objective To investigate the mechanical properties of carbon-fiber-reinforced polyether-ether-ketone(CF/PEEK) composite materials and to evaluate the biocompatibility and bioactivity of CF/PEEK after implanted into mandibula.  Methods 27 adult Japanese white rabbits were randomly divided into experimental group (n=9) and control group (n=18),CF/PEEK was implanted into bilateral mandibula of the rabbits in experimental group,autologous bone was implanted into left mandibula of the rabbits in  positive control group(n=9) while the right sides were taken as blank control group(n=9) without implants.The materials-bone interface of samples were collected to make the observation of gross， imaging and histology and to analyze the  histological score after 8,12,and 16 weeks. Results All the animals and  no infection  had secondary fractures.Obviously callus formation was observed on the edge of the defect in both experimental group and positive control group in the imaging observation,while the rabbits in blank control group had  no obvious change. Compared with 8 weeks after operation，the score evaluation of local cellular immune response showed significant decreasing in  12 weeks after operation(P<0.01)，the data was the  minimum in 16 weeks（P<0.05）.At the same time,the score evaluation of local cellular immune response in three groups showed no significant difference (P> 0.05).Trabecular bone and osteoblasts could be observed in the bone defect of both experimental group and positive control group after 16 weeks,and blank control group was filled with fibrous connective tissue.The osteogenesis rates  in experimental group and positive control group were increased with the prolongation of time,and they were better than that in blank control  group(P<0.01). Conclusion CF/PEEK material has good  mechanical property,biocompatibility and bioactivity;and it has light immune response after implantation.

Abstract:Objective To observe the distribution of oligodexynucleotide (ODN) MT01 in main organ tissues  of the rats at different time points and to discuss the regularity of the distribution of MT01 preliminarily.Methods 60 male Wistar rats were randomly divided into experimental group(n=30) and control group(n=30).The rats in experimental group was locally injected  with Cy5 labeled MT01 in gingival mucosa,whereas the rats in control group were injected  with MTO1.The  samples of rat lung,liver spleen,kidney,heart,and brain tissues  were collected at 15 min,1 h,4 h,8 h,16 h,1 d,2 d,3 d,4 d,and 5 d after injection, and the distribution of MT01 fluorescence was observed by laser scanning confocal microscope.The ratio of fluorescence positive cells indicated the amount of MT01 that had been taken up by different organs.Results No positive fluorescence cells were observed in control group.Whereas，in experimental group ，the positive fluorescence cells were detected in the tissue samples of lung,liver,spleen and kidney but not in the tissue samples of heart and brain.The positive fluorescence cells  distributed focally in kidney tissue and presented primarily in the cytoplasm of renal tubular epithelial cells.The ratios of positive fluorescence cells changed regularly with time in liver,spleen and kidney tissues and the highest level was detected at 4，3 and 4 d after injection.No distinct regularity of the ratio of positive fluorescence cells was observed in lung tissue.Conclusion MT01 can be taken up by liver，spleen and lung tissue and prima
rily by kidney with regularity in distribution.

Abstract:Objective To study the biocompatibility of silk fibroin/poly L-lactic acid(SF/PLLA) non-woven network,a kind of new composite tissue engineering nanomaterials,and to explore its possibility as the biological implant materials.Methods  The PLLA non-woven network was prepared by electrostatic spinning.Physiological saline as control,the leaching solution was prepared and injected into the mice,then the mice were observed for 2 weeks.The materials were implanted into the back of the mice,and 3-0 suture was used as control.Tissues were collected at 1,2,3,and 4 weeks after operation,dyed by HE staining and then the  photos were taken.The tissue reactions in experimental group and control group were observed.The rabbit knee joint cartilage cells were cultured,and then subculture cells were seeded to the surface of materials.After cultured in vitro,the adhesion and growth of the cells were observed with  inverted optical microscope.The bioactivities of the rabbit knee joint cartilage cells in negative control group(DMEM culture media),experimental group(DMEM containing materials) and positive control group(DMEM containing phenol solution) were determined by MTT assay after cocultured for 24 and 48 h.Results After injection，the body status of the mice in experimental group was the same to the control group.There were little fibroblasts was  and a little of lymphocytes and macrophage cells in the materials which were implanted into the back of the mice at the beginning.Then the number of the fibroblasts was increased,but the number of the lymphocytes  and macrophage cells did not change obviously.The materials degraded slowly,and the material degraded  obviously at 4 weeks.The inflammation of tissue around the material reduced  gradually from the  2nd week.The inflammation of tissue around the material was the same to the suture,and sometimes was slighter than the suture.After sed for  24 h,there were cells attaching to the fibers of the material.More and more cells attached to the fibers.The reasult of MTT assay showed that the cytotoxicities in experimental groups were all on Level Ⅰ at 24 and 48 h.Except for positive control group,the A values were increased in other groups with the extended response time.At the same time,there was no significant difference in cytotoxicity between experimental group and negative control group(P>0.05) and the A value in experimental group was higher than that in positive control group(P<0.01).Conclusion The SF/PLLA non-woven network scaffold material has good biological compatibility and safety,it could be used as implant material in tissue engineering.

Abstract:Objective To construct  in the  extracellular matrix metalloproteinase inducer (EMMPRIN) glycosylation single point mutation plasmid,and to explore its relationship with  tumor cell proliferation.Methods PCR point mutantion technology was used to construct the mutantion plasimid of EMMPRIN glycosylation single point.After successful mutation,the function of mutantion plasmids were detected.Western blotting was used to detect the expression of EMMPRIN protein,immunofluorescence method was used to detemine the morphological changes of the cells, and MTT assay was performed to detect the relationship between mutantion pasmid and tumor cell proliferation.Results Confirmed by restriction enzyme digestion and sequencing,the 44th,the 152th, and the 186th Asn were successfully mutated to Gln in the sequence of EMMPRIN; EMMPRIN/GFP(N44Q),EMMPRIN/GFP(N152Q), and EMMPRIN/ GFP(N186Q) glycosylation single point mutation plasmids were constructed.Compared with wild-type,thel morphology of the cells was significantly changed,the core division of mutant-type cells was significantly reduced,the number of filopodia was reduced.The results of MTT assay showed that the survival rate of the cells in wild-type group were significantly increased compared with  control group (P<0.05);the survival rates of the cells in EMMPRIN(N44Q) group,EMMPRIN(N152Q) group  and EMMPRIN(N186Q) were significantly decreased compared with wild-type group(P<0.05).Conclusion Mutant-type EMMPRIN can inhibit the proliferation of tumor cells; with the duration increasing,the inhibitory effect is weakened.There is a correlation between EMMPRIN glycosylation and proliferation of tumor cells.

Objective To explore the protective effects of puerarin on the colon tissue of the mice wi
th chronic alcoholism through observing the changes of quantity and morphology of
interstitial cells of Cajal （ICC）  and the expression of c-kit protein in
colon tissue of chronic alcoholism mice.Methods 24 healthy BALB/C mice were randomly divided into saline control group (SC group),chronic alcohol
ic intoxication group(CAI group) and puerarin pretreatment group(PUE group).
The models of chronic alcoholism mice were established in CAI group and PUE group.
The intestinal transmission rate was tested by measuring the Indian ink advancing length; immunofluorescence and transmission electron microscope (TEM) were applied to detect the distribution,quantity and ultrastructure of ICC; Western blotting method was used to analyze the C-kit protein expression.
Results The intestinal transmission rate in PUE group was significant higher than that in CAI group(P<0.05),but was still lower than that in SC group (P<0.05); compared with CAI group,the number of ICC in PUE group was increased (P<0.05),but there was no significant difference between SC group and PUE group（P>0.05）.Compared with SC group,the ultrastructure of ICC  in CAI group had obvious changes, the organelles were decreased, and the mitochondrion was swelling.But those changes were reversed in PUE group and the  number of mitochondria was increased.The expression of C-kit protein in PUE tissue was higher than that in CAI group (P<0.05),but thers no significant difference compared with SC group.Conclusion Puerarin has a repair or reverse effect on the changes of the number,ultrastructure of ICC and C-kit protein expression caused by alcohol.

Objective To study the influence of hydrogen sulfide (H2S) on the  mitochondrial function of pulmonary artery smooth muscle cells of the  rats with high pulm
onary blood flow pulmonary hypertension,and to clarify the function and its mechanism in the occurrence of  high pulmonary blood flow pulmonary hypertension.Methods A total of 27 rats were randomly divided sham operation group,operation group,and operation+sodium hydrosulfide (NaHS) group (n=9).The pulmonary hypertension rat model was built by left lung resection.After fed for 35 d,the mean pulmonary artery pressure (mPAP),ratio of right ventricle / body weight (RV / BW),and ratio of right ventricle /(left ventricle+septum) ［RV /(LV + S)］ of the rats in three groups were measured.The plasma H2S levels and the CSE activities in pulmonary artery tissue of the rats were detected;the activities of total mitochondrial ATP enzyme,superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) levels were determined;the ultrastructure of pulmonary artery smooth muscle mitochondria was observed through transmission electron microscope.Results  Compared with sham operation group,the plasma H2S level and the CSE activity in p
ulmonary artery tissue of the rats in operation group  were decreased(P<0.01);the mitochondrial membrane of pulmonary tissue was swelling,and the mitochondrial activity was decreased(P<0.01);the mitochondrial ATP enzyme,SOD and GSH-Px activities were significantly decreased,and the MDA level was significantly increased (P<0.01).Compared with operation group,the H2S level in  plasma and the activity of CSE in pulmonary tissue of the rats in operation + NaHS group were increased(P<0.01);the mitochondrial membrane swelling was reduced,and the vitality was restored;the ATP enzyme,GSH-Px,and SOD level in pulmonary tissue were significantly increased (P<0.01),and the MDA level was significantly reduced(P<0.01).Conclusion H2S can enhance the activities of mitochondrial ATP enzyme,GSH-Px,and SOD,and decrease the mitochondrial lipid peroxidation level,thus it plays a protective effect on  rat pulmonary artery smooth muscle.

Objective To investigate the protective effect of nimodipine on neuron of the rats with focal cerebral ischemia-reperfusion injury and the expressions of Bax and Bcl-2,and to clarify their mechanisms. Methods The focal cerebral-ischemia reperfusion model was induced by the middle cerebral artery occlusion(MCAO)
 method. 30 male Wistar rats were randomly divided into sham operation,model,and nimodipine  groups(n=10). The neurological deficit score was performed after 2 h ischemia following 2 h reperfusion. The infarction was observed by TUNEL staining and the expressions of Bax and Bcl-2 were detected by SP immunohistochemistry method. Results Compared with model group,the number of apoptotic cells of the rats in nimodipine group was significantly decreased(P<0.05),the expression of Bax  was significantly decreased (P<0.05),and the  Bcl-2 expression was increased significantly(P<0.05).The morphological examination showed that the neurons of the rats in model group had  serious necrosis and edema while  the number of  dead cells in nimodipine treatment group  was reduced and the edema was improved.Conclusion Nimodipine has a protective effect on brain tissue of the rats with focal cerebral ischemia-reperfusion injury，which is
closely related to the down-regulation  of Bax and up-regulation of Bcl-2 and inhibition of the apoptosis of neuron.

Objective  To observe the lipid regulation  role of electroacupuncture intervention in the rats with non-alcoholic fatty liver disease(NAFLD) induced with high fat and cholesteol forage,and to clarify the regulation mechanism of  serum retinol binding protein 4(RBP4) level and liver X receptor a (LXR-α) and sterol regulatory element binding protein-1c(SREBP-1c) expressions. Methods 44 female SD rats were fed for 7 d to adapt the environment and were randomly divided into normal group,model group,Dongbaogantai group,electroacupuncture group; 11 rats in each group. The rats in normal group got routine feeding,and
the others were fed with high fat and high cholesterol forage. After 8 weeks,the models were established,the rats in electroacupuncture group
  were treated with electroacupuncture method (1.5-2.0 Hz,D.-D.wave,9V,1-3 mA)  in “Ganshu”,“Pishu”,“Geshu” for 15 min,once a day,lasted for 28 d. The changes of fasting blood-glucose(FBG),serum free fatty acids(FFA) and liver tissue homogenate triglyceride(TG) and total cholesterol(TC) levels of the rats in various groups  were tested,and enzyme-linked immunosorbent (ELISA) was used to determine the serum RBP4 levels,and Western blotting method was used to detect the  LXR-α and SREBP-1c protein expression levels in rat liver tissue. Results Compared with normal group,the FBG,serum FFA, TG and TC levels in liver tissue  homogenate and serum RBP4 level of the rats in model group were increased (P<0.01);the LXR-α and SREBP-1c protein expression levels were also increased (P<0.01). Compared with model group,the FBG,serum FFA, the TG and TC levels  in liver tissue homogenate and serum RBP4 levels of the rats in
electroacupuncture group and Dongbaogantai group were decreased (P<0.01); the LXR-α and SREBP-1c protein expression levels were also decreased (P<0.
05 or P<0.01). Conclusion Electroacupuncture method in “Fenglong”,“Zusanli”,“Sanyinjiao” can reduce the serum RBP4 level,regulate the lipid metabolism,and improve the lipid deposition of the NAFLD rats;they  have obvious therapeutic effect on NAFLD,and its mechanism may be related to inhibiting the increasing of LXR-α and SREBP-1c protein expressions in liver tissue.

Objective To explore the influence of salinomycin in the  growth,apoptosis and invasion of human bladder cancer 5637 cells,and to clarify its possible mechanism.Methods The human bladder cancer 5637 cells cultured in vitro at logarithmic growth phase were divided into control group and different doses of salinomycin(15,30 and 60 μmol?L-1) groups.The inhibitory rate of the growth of 5637 cells in various groups  was measured by MTT assay.Flow cytometry was used to detect the apoptotic rates of 5637 cells in various groups.The invasiveness of 5637 cells was tested by Matrigel Invasion Assay.The  expression levels of β-catenin protein in 5637 cells in various groups were determined by Western blotting method. Results Compared with control group,the inhibitory rates of
 growth of human bladder cancer 5637 cells in different doses of salinomycin groups were increased significantly(P<0.05); the  apoptotic rates were   increased(P<0.05).the number of cells passed the Matrigel was decreased(P<0.05),and the expression level of β-catenin protein was decreased(P<0.05).Compared with low dose of salinomycin group, the inhibitory rate of growth of 5637 cells in high dose of salinomycin group was increased(Ｐ<0.05);the apoptotic rate was increased(P<0.05)，the number of cells passed the Matrigel was decreased(P<0.05),and the expression levels of β-catenin protein was decreased(P<0.05).
Conclusion Salinomycin can inhibit the growth of 5637 cells significantly,increase the apoptosis,and decrease the cell invasion; the inhibitory effect may act by inhibiting the Wnt/β-catenin pathway.

Objective To explore the therapeutic effect of phenylethanoid glycosides on cyclophosphamide (CTX)-induced dyszoospermia in mice and to preliminary elucidate the mechanisms involved in the process. Methods Phenylethanoid glycoside was extracted by ethanol extraction. Forty male BALB/C mice were randomly divided into control group,model group,low dose of  phenylethanoid glycosides group (50 mg/kg^-1) and high dose of phenylethanoid glycosides group (100 mg/kg^-1). Except control group,the dyszoospermia mouse model was established by peritoneal injection of CTX at the daily dose of 80 mg?kg-1,once daily for  successive 5 d. After modeling,phenylethanoid glycosides were intragastrically administered at corresponding doses to each phenylethanoid glycosides group. Equal volume of
 normal saline was given to the mice in control group and model group by gastrogavage. All the medication was performed once daily for successive 30  d. The testis tissue was  obtained 24 h after the last intragastric administration. The level of testosterone in the testis tissue homogenate was determined by enzyme linked immunosorbent assay. The sperm counts,the motility rates,and the teratospermia rates in various groups were compared. The morphological changes of the testis tissue were observed using HE staining. Results Compared with control group,the sperm count and the motility rate were decreased,the teratospermia rate was increased,and the testosterone level in the testis tissue homogenate was decreased in model group(P<0.01). Compared with model group,the sperm counts and the motility rates were increased,the teratospermia rates were decreased,and the testosterore  levels in the testis tissue homogenate were increased in phenylethanoid glycosides groups (P<0.05 or P<0.01). The histological results showed atrophy and degeneration of seminiferous tuble, thicker seminiferous epithelium  and azoospermic lumina in  model group; the number of seminiferous epithelial layers was increased and the seminiferous cells orderly arranged,and many sperms were found in the tubules in  phenylethanoid glycosides groups. Conclusion Phenylethanoid glycosides has obviously therapeutical effect on CTX-induced dyszoospermia in mice,and its mechanisms might be correlated with recovering the testosterone level.


Objective To explore the influence of Akt inhibitor MK2206 in the proliferation and apoptosis of tongue squamous cell carcinoma TCA-8113 cells，and to clarify the possible mechanism.Methods The tongue squamous carcinoma TCA - 8113 cells at the logarithmic phase  were randomly divided into control group and 1,5,25,125,250 nmol/L MK2206 groups.The inhibitory rate of proliferation of TCA-8113 cells was detected with MTT method,and the a
poptotic rate of TCA-8113 cells was determined with flow cytometry(FCM),and the expressions of caspase-9,Bad,GSK-3β,p-Akt and T-Akt proteins
in the  TCA-8113 cells were detected with Western blotting method.Results The IC50 of tongue squamous cell carcinoma TCA - 8113 cells after treated with MK2206 for 12,24,and 36 h were (112.54±1.67),(79.67±2.01),and (33.33±1.98) nmol/L.The FCM results showed that the apoptotic rates of TCA-8113 cells after treated with 1,5,25,125, and 250 nmol/L MK2206 for 12 h  were （14.2±0.74）%,（19.3±0.45）%,（35.1±0.45）%,（39.6±0.48）% and （52.1±0.19）%；there were significant differences compared with  control group(P<0.01).The Western blotting method results showed that  the expressions of p-Akt,Bad and  GSK-3β were decreased with the increasingof dose and time of MK2206; compared with  the  β-actin in  control group,the bands got darken;the expression level of caspase-9 was increased,compared with the β-actin in control group,the bands got darken;the  T-Akt protein expression did not change significantly;compared with the β-actin in control group, the  color of bands had  no significant difference. Conclusion Akt inhibitor MK2206 can inhibit the proliferation of tongue squamou
s cell carcinoma TCA-8113 cells and induce apoptosis.

Objective To investigate the inhibitory effect of  siRNA targeting casein kinase 2 （CK2α） gene on the growth  of HCT116 cells and to clarify its mechanism.
Methods CK2α-siRNA sequence was designed according to mRNA sequence of CK2α.The in vitro cultured HCT116 cells were divided
 into normal control group(without transfection),negative control group(transfected with siRNA) and CK2α-siRNA group(transfected with CK2α-siRNA ),and the HCT116 cells were transfected with Lipofectamine 2000.The expression levels  of CK2α,cyclin H,P53,and P21 proteins in the HCT116 cells were detected by Western blottting method,and  the proliferation activities of the HCT116 cells were detected by MTT method,and the cell cycle was measured by flow cytometry.
Results Compared with negative control group,the expression levels of CK2α,and cyclin H proteins in CK2α-siRNA group were decreased（P<0.01）;the expression level of P53 protein had no change dramatically（P>0.05）,and the expression level of P21 protein was increased significantly（P<0.01）.Compared with negative control group,the survival rate in CK2α-siRNA group was decreased markedly 48 and 72 h after transfection detected by MTT method（P<0.01）.Fl
ow cytometry analysis showed the percent of the  cells at  G1 phase in CK2α-siRNA group was significantly higher than that in negative control
group and the percent of the cells at S phase  in CK2α-siRNA group was lower than that in negative control group（P<0.01）,and the cell cycle was arrested at G1
 phase.Conclusion siRNA targeting CK2α can inhibit the proliferation of HCT116 cells and induce the arrest of G1 phase,which may be associated with inhibiting the expression of cyclin H and recovering the P53 activity after silencing CK2α.

Objective To explore the risk factors of vascular cognitive impairment (VCI) among Chinese population,and to clarify the scientific evidences for further prevention and treatment.Methods PubMed,CNKI,CBM,VIP and Wangfang databases (from 2002.1 to 2013.1) were searched to collect case-control studies or cohort studies studying risk factors of VCI among Chinese population.Meta-analysis was performed to calculate combined odds ratio (OR) or mean difference (MD) and its 95% confidence interval (95%CI).Results A total of 42 proper papers involving 3 282 cases and 7 815 controls were included in the review.For categorical variables,pooled OR and its 95%CI were as follows:hypertension　2.56(2.03－3.21),hyperlipidemia1.79(1.39－2.30),hyperglycemia 2.46(1.90－3.19),Leukoaraiosis 5.46(2.60－11.46),cerebral infraction multiple foci 4.39(2.61－7.38),stroke history3.79(2.35－6.11),left hemisphere lesions 2.13(1.42－3.20),smoking 1.51(1.08－2.11),drinking 0.99(0.73－1.36),basal ganglia lesions 2.15(1.55－2.99),thalamus lesions 2.34(1.57－3.47);for continuous variables,MD and its 95%CI were as follows:level of TG 0.35(0.15－0.55),level of TC 0.44(－0.16－1.04),level of folic acid －4.10(－5.50－ －2.69),vitamin B12  －130.44(－225.46－ －35.41). Conclusion Except for drinking and level of TC,hypertension,hyperlipidemia,hyperglycemia,leukoaraiosis,cerebral infraction multiple foci,stroke history,left hemisphere lesions,smoking,basal ganglia lesions,thalamus lesions,high level of TG,low level of folic acid and vitamin B12 might be the risk factors of VCI among Chinese population.

Objective To investigate the dynamic changes and prognosis of thyroid function in the patients with chronic hepatitis C (CHC) after antiviral treatment,and to clarify the  influence of  baseline factors in the changes of thyroid function.Methods 243 CHC patients with normal baseline thyroid function
 were enrolled.All patients were treated with  IFN-alpha-2b(IFN-α2b) combined with ribavirin for 48 weeks.The thyroid function and serum HCV RNA level were assessed at 12,24,36,48,60 and 72 weeks. According to the changes in thyroid function after treatment,the patients were divided into continued normal,subclinical hypothyroidism,hypothyroidism and hyperthyroidism groups.The regularity of the changes of thyroid function of the patients in various groups were observed.
Results Among 243 CHC patients,82(33.7%) patients had thyroid dysfunction. The  prevalence of subclinical hypothyroidism,hypothyroidism and hyperthyroidism were 20.9%(51/243),5.3%(13/243)  and 7.4%(18/243),  respectively. At the end of 72 weeks,there were 32(39.0%) patients suffering from subclinical hypothyroidism，12(14.6%)  patients with  hypothyroidism  and 7(8.5%)  patients with hyperthyroidism  rehabilitated.6（7.3%）　patients suffering from hypothyroidism turned to subclinical hypothyroidism,and 3(3.7%） patients suffering from hyperthyroidism turned to subclinical hypothyroidism.19（23.2%） patients had no significant change,they performed for continued subclinical hypothyroidism (1,1.2%),hypothyroidism (13,15.9%） and hyperthyroidism (5,6.1%).In addition,3 (3.7%) patients with hyperthyroidism turned to hypothyroidism.  An increased risk for hypothyroidism was found in female patients compared with males (P<0.05);the average age of the patients with hyperthyroidism was lower than those of the patients with hypothyroidism,subclinical hypothyroidism and continued normal (P<0.05);the baseline levels of GGT in the patients with hyperthyroidism and hypothyroidism were lower than those of the patients with subclinical hypothyroidism and continued normal(P<0.05).The ratio of the patients with HCV 2a to the patients with hypertyroidism was higher than those of the patients with hypothyroidism,subclinical hypothyroidism and continued normal(P<0.05).Conclusion Thyroid function in the CHC patients can be affected by antiviral treatment.Gender,age,liver function,genotype of HCV  are influencing  factors for the changes of thyroid function.
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Objective To compare the biochemical and  immunological parameters and histopathological characteristics  in the patients with autoimmune hepatitis
 (AIH),biliary cirrhosis (PBC) and AIH/PBC overlap syndrome,and to provide basis for working out the reference standard of clinical diagnosis and reasonable treatment. Methods 135 cases of autoimmune liver disease patients were selected,including 49 cases of AIH patients,43 cases of PBC patients,and 43 cases of AIH/PBC overlap syndrome  patients.The biochemical,immunological parameters and histopathological changes of the patients were dete
cted.The patients  with  AIH / PBC overlap syndrome were treated with different therapy methods including ursodesoxycholic acid(UDCA) treatment,prednisone treatment and combination treatment with UDCA and prednisone,and   the effectiveness of different treatment programs were evaluated.Results The activities  of  GGT and ALP and IgM level of the patients in the AIH/PBC group were significantly higher than those in   AIH group,there were significant differences (P<0.05);the activities of ALT and AST and IgG level of the patients in  AIH/PBC group was significantly higher than those in  PBC group,there were significant differences (P<0.05).The detection rates of AMA and AMA M2 of the patients in AIH/PBC group were higher than those in AIH group,there were significant differences (P<0.05);the detection rates of ANA and SMA of the patients in AIH/PBC group were higher than those in PBC group,there were significant differences (P<0.05).There were high incidence of piecemeal necrosis (100.0%),liver cell rosette-like changes (83.72%) and bile duct lesions (69.77%) of the patients in AIH/PBC group.The effective percentage in combination therapy group was 85.7% which was significantly higher than those in various drug alone groups (P<0.05).Conclusion The changes of biochemical and immunological indicators  and pathological features of the patients with   AIH/PBC overlap syndrome are in  combination with the particular indicators of AIH and PBC which  would provide the diagnostic basis for AIH/PBC overlap syndrome.The combined therapy for AIH/PBC overlap syndrome is effective and should be popularized.

Objective To detect the expression levels of urokinase-type plasminogen activator(uPA)，matrix metalloproteinase-3 (MMP-3),MMP-9,MMP-13 and
MMP-14 in the patients with osteoarthritis(OA) before and after arthroscopic debridement,and to explore the influence of arthroscopic debridement in the expressions of uPA,MMP-3,MMP-9,MMP-13,and MMP-14.Methods 420 cases of synovial fluid from knee OA patients undergoing arthroscopic debridement  were obtained before operation.After six months follow-up,350 cases of synovial fluid samples were obtained and according to inclusion and exclusion criteria,228 synovial fluid were selected to analyze.The expression levels of uPA,MMP-3,MMP-9,MMP-13, and MMP-14 were measured by ELISA assay.Pain intensity of these patients before operation and  six months after operation were recorded using the Visual Analogue Scale/Score(VAS).The differences of the expression levels of uPA,MMP-3,MMP-9,MMP-13,and MMP-14 between before operation and after operation were compared.The relationship between the expression levels of uPA,and MMP-3,MMP-9,MMP-13,MMP-14 and VAS was analyzed with Spearman analysis.Results All the patients were followed up for 36.5 months.Compared with before operation,the expression levels of uPA and MMP-3  in the synovial fluid of the patients  after arthroscopic debridement  were significantly decreased(P<0.01),the expression levels of MMP-9  and  MMP-13 were also decreased (P<0.05）,but  the MMP-14 expression level showed no significant change. The expression levels of  uPA,MMP-3,MMP-9,MMP-13,MMP-14  were positively associated with VAS before arthroscopic debridement r=0.361,r=0.417,r=0.136,r=0.514,r=0.156,P<0.05);uPA and MMP-3 were positively correlated with VAS  after arthroscopic debridement（r=0.981，r=0.831，P<0.01），as well as the expression level of MMP-13 and VAS,but there were no significant differences between the expression levels of MMP-9,MMP-14 and VAS.Conclusion The decreased levels of uPA, MMP-3 and MMP-13 in synovial fluid may contribute to the pain-relief effects of arthroscopic debridement.
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To explore the association between urinary lactate/creatinine ratio and neonatal asphyxia,and to clarify the application value of urinary lactate/creatinine ratio  in forecasting the hypoxie-ischemic encephalopathy （HIE） in neonates.Methods Using case-control study design method,40 cases of neonatal asphyxia infants were selected as  asphyctic group and 40 healthy infants were used as control group.The  urinary lactate/creatinine ratio in the first day after birth,urinary N-Acetyl-β-D-glucosaminidase (NAG)/creatinine and the score of Apgar of 80 infants were detected.The relationship between the urinary lactate/creatinine ratio,urinary NAG/creatinine ratio,as well as the score of Apgar and HIE were analyzed and compared in asphyctic group and control group.Results The Apgar scores at 1 and 5 min in asphyctic group were significantly lower than those in control group (P<0.01). The urinary lactate/creatinine ratio and urinary NAG/creatinine ratio  in asphyctic group 1d after birth were significantly higher than those in control group(P<0.01). There was  significantly negative correlation between the urinary lactate/creatinine ratio and the   1-min  and 5-min Apgar scores(r=－0.636，P<0.001; r=－0.883，P<0.01),but there was a significantly positive correlation between the urinary lactate/creatinine ratio and urinary NAG/creatinine  ratio(r=0.433，P<0.01).Conclusion The urinary lactate/creatinine ratio  may have decisive roles in prognosis evaluation of neonatal asphyxia,and so as the important foundation for the prediction of HIE.
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Abstract:Objective To investigate the expressions of apoptosis stimulating protein of P53 (ASPP) family in colorectal carcinoma tissue and to explore their relationship with the clinicopathological characteristics of colorectal carcinoma,and to clarify the effect of ASPP  family in the development of colorectal carcinoma.
Methods 45 cases of colorectal carcinoma tissue and 20　cases of healthy controls were selected.Among 45 cases of colorectal carcinoma tissue,there were
 11 cases of well differentiated colorectal carcinoma,21 cases of moderately differentiated colorectal carcinoma,and 13 cases of poorly differentiated color
ectal carcinoma;7 cases of T1 stage colorectal carcinoma,8 cases of T2 stage colorectal carcinoma,25 cases of T3 stage colorectal carcinoma,and 5 cases of T4
 stage colorectal carcinoma;19 cases of N1 stage with lymph node metastasis,26 cases of N0 stage without lymph node metatasis.The expressions of ASPP1,ASPP2,and iASPP in 45 cases of colorectal carcinoma tissue  and 20 cases of normal colon tissue were detected by immunohitochemistry SP method,and the correlations between the expressions of ASPP  family and the pathologic typing,infiltrative depth,and lymph node metastasis of colorectal carcinoma were analyzed.Results ①The immunohitochemical staining results showed that the ASPP  family members expressed in colorectal carcinoma tissue and normal colon tissue,and there were no significant differences in ASPP1 and ASPP2 positive rates between colorectal carcinoma tissue and normal colon tissue (P>0.05);the positive expression rate of iASPP in colon cancer tissue was higher than that in normal colon tissue (P<0.01).② The expression of ASPP1 in colon caner tissue had no correlation with the differentiation degree of tumor cells (rs =0.163,P>0.05);the expression of ASPP2 positive rate was decreased when the differentiation degree of tumor cells reduced,they had positive correlation (rs =0.454,P=0.002)；the expression of iASPP in colon cancer tissue had no correlation with the differentiation degree of tumor cells (rs =－0.171,P>0.05).③ The expression of ASPP1 in colon caner tissue had no correlation with the infiltrative depth of tumor (rs =－0.268,P>0.05);the expression of ASPP2 positive rate was decreased when the tumor infiltrative depth increased,they had negative correlation (rs=－0.348,P<0.05);the expression of iASPP in colonic carcinoma caner tissue had no correlation with the infiltrative depth of tumor (rs =0.231,P>0.05).④The expressions of ASPP1,ASPP2,and iASPP in colon caner tissue had no correlation with lymph node metastasis (rs =0.089,rs =0.044,rs =0.210,P>0.05).Conclusion The expression levels of iASPP in colon cancer and normal colon tissues are different,it may be useful for the diagnosis,differential diagnosis and evaluation in benign and malignant colorectal diseases.   The expression of iASPP is negatively correlated with the pathologic typing and neoplasm staging of colorectal carcinoma,it indicates that iASPP can be used as a indicator in judging the prognosis of colorectal carcinoma.

Objective To investigate the expressions of tissue factor pathway inhibitor-2 (TFPI-2) and matrix metalloproteinase-9 (MMP-9) in esophageal squamous c
ell carcinoma（ESCC）tissue and their relationship with vasculogenic mimicry (VM).Methods 162 cases of esophageal squamous cell carcinoma tissues
 were collected.CD34/periodic acid-schiff double staining was performed to observe the distribution of VM in ESCC tissue,and immunohistochemical staining was used to detect the expressions of  TFPI-2 and MMP-9 in ESCC tissue.The relationship between VM  and the clinicopathologic parameters of ESCC,the expressions of TFPI-2 and MMP-9 were analyzed.Results The positive rate of VM in  ESCC tissue was 20.37%.The positive rate of VM in poorly differentiated group(40.38%) was significantly higher than those in moderately differentiated group(11.76%) and well differentiated group(7.14%)（χ2=20.915，P<0.01）.The positive rate of VM in TNM Ⅰ-Ⅱ (11.59%) group  was significantly lower than that in TNM Ⅲ group(26.88%)（χ2=5.707，P=0.017）.The positive rate of TFPI-2 in VM(+) group(33.34%) was significantly higher than that in VM(-) group(6.45%) （χ2=4.582，P=0.032） in poorly differentiated ESCC. The positive rate of MMP-9 in VM(+) group(78.79%) was significantly higher than that in VM(-) group(44.96%) （χ2=12.05，P=0.001）. The expression level of TFPI-2 in poorly differentiated group was positively correlated with  VM（r=0.166，P=0.032),and the expression level of MMP-9 was positively correlated with the VM（r=0.183,P=0.018）.The five-year survival rate in VM(－) group was significant higher than that in VM(+) group(χ2=22.84，P<0.001).Conclusion VM exists in ESCC tissue,especially in poorly differentiated and advanced ESCC tissue.VM is related to poor  prognostis of  ESCC.TFPI-2 and MMP-9 might involve in the formation of VM in ESCC.

Objective By comparing the detection rates of subclinical thyroid diseases in women(pregnant,lactating and child-bearing age) between iodine deficient regions (supplied iodized salt) and water-borne iodine excess regions(consumed non-iodized salt),and to find the different prevalence of subclinical thyroid disease between two regions under their different iodine source and iodine levels,and to provide reference for screening susceptible population with iodine-related thyroid diseases.Methods The iodine deficiency but salt iodine surpplying regions were selected from six provinces in our country,the local people who were pregnant women,lactating women and 18 to 45 years old women of child-bearing age,a total of 991 cases were investigated.The iodine nutrition levels of the pregnant women were grouped by <150,150-249,and ≥500 μg?L-1;the iodine nutrition levels of the lactating women were grouped by <100 and ≥100 μg?L-1.The high water-iodine regions in Shanxi Province were selected,and according to the water-iodine levels 50－99,100－149,149－299,and more than 300  μg?L-1 four regions were selected; 20 cases of three kinds of people mentioned above were selected in each region,a total of 241 cases.The blood and urine samples were collected,and the serological thyroid function indexes were detected by chemiluminescence immunoassay method or radioimmunity method,and the urine-iodine was detected with cerium catalytic spectrophotometric method.Results In iodine deficient regions and water-borne iodine excess regions,the concurrence rates of subclinical hypothyroidism(hypothyroidism for short) and thyroid antibody positive of women were 2.32% and 4.98%,respectively;accounting to about 1/3 to 1/2 of those subclinical hypothyroidism population.The prevalence rates of subclinical thyroid diseases in the women population between the two regions were 27.55% and 34.85%,respectively;nearly accounting for 1/3 of the women population.The subclinical hypothyroidism detection rates of the three populations in high water iodine regions were significantly higher than those in iodine deficient regions(P<0.05).The lactating women’s detection rates of thyroid antibody positive and subclinical hypothyroidism with antibody positive in high water iodine regions were significantly higher than those in iodine deficient regions(P<0.05).With different iodine sources,when took appropriate iodine,there was no statistical difference of the detection rates of subclinical thyroid diseases among three kinds of women(P>0.05).With the increase of iodine exposure levels,the prevalence of women who suffered from subclinical hypothyroidism and thyroid antibody positive was increased,the coincidence rate was  also increased.The detection rates of low T4 concentration and total subclinical thyroid disease of pregnant women in iodine nutrition <100  μg.L^-1group were significantly higher than those in  iodine nutrition 250－499  μg.L^-1 group (P<0.05).The detection rates  of low T4 concentration and total subclinical thyroid disease of lactating women in iodine nutrition <100  μg.L^-1group were significantly higher than those in  iodine nutrition >100  μg.L^-1group (P<0.05).Conclusion When the iodine intake is appropriate,iodine intakes from salt or from water have no effect on subclinical thyroid diseases.When the iodine intake increases,the prevalence of subclinical thyroid diseases will increase too.

Objective To study the ages at natural menopause of the women in Jilin Province,and to illustrate its influencing factors among the women in Jilin Province.
Methods Through multistage stratified cluster random sampling method，23 050 people aged from 18 to 79 years were drew from nine states(a total of 32 areas) of Jilin province.The data of these residents were collected with the questionnaire and physical examinations by face-to-face interview.The number of selected female sample was 11 098.Finally,4 881 postmenopausal women  were selected.Complex weighted computation was used to estimate the ages at natural menopause.One-way ANOVA was used to compare the ages atnatural menopause of the women with different birth years.Multiple linear regression analysis were used to examine the influencing factors of the ages at natural menopause.Results The mean and median ages at natural menopause were　(49.11±4.19) years and 50.00 years, respectively.There were 4 881 cases of   postmenopausal women,among them  the women with age at natural menopause＜40 years,40 year≤age at natural menopause≤45 years,46 years≤ age at natural menopause≤53 years,age at natural menopause ≥54 years and age at natural menopause missing accounted for 2.27%(111 cases),13.17%(643 cases),71.97% (3 513 cases),11.74% (573 cases), and 0.85%（41 cases),respectively. Converted to birth years by age,70-79 years old was 1933-1942 birth years,60-69 years old was 1943-1952 birth years and 57-59 years old was 1953-1955 birth years.The age at natural menopause in Jilin province was statistically significant among the women with different birth years（F=21.178,P<0.001）.By SNK-q test among three different birth year groups,the age at natural menopause was different between any two groups among three different birth year groups and the ages at natural menopause of 1953-1955 birth year group,1943-1952 birth year group and 1943-1952 birth year group were  50.38 years,49.51 years and 48.81 years.The age at natural menopause in urban of Jilin province was statistically significant among the women with different birth years（F=16.633，P<0.001）.By SNK-q test among three different birth year groups,the age at natural menopause was different between any two groups among three different birth year groups and the ages at natural menopause of 1953-1955 birth year group,1943-1952 birth year group and 1943-1952 birth year group were  50.77 years,49.73 years, and 48.85 years,respectively.The age at natural menopause in rural of Jilin province was statistically significant among the women with different birth years（F=7.400，P=0.001）.By SNK-q test among three different birth year groups,the age at natural menopause was different between 1953-1955 birth year group and the other two groups and the ages at natural menopause of 1953-1955 birth year group,1943-1952 birth year group and 1943-1952 birth year group were  50.09 years,49.33 years, and 48.74 years,respectively.The multiple linear regression results indicated that BMI and exercise were positively correlated with the age at natural menopause,but smoking and mental health evaluation were negatively.Consumption frequency of vegetables, fruits, bean products, and meat was no correlated with the age at natural menopause.Conclusion The differences of the ages at natural menopause between the women with  different birth years are statistically significant in Jilin Province;BMI,smoking,exercise, and mental health are the influencing factors of the age at natural menopause.

Objective  To estimate the dietary diversity,overweight and obesity of the rural adults aged 18-65 years in Jilin Province by diet diversity score(DDS),
and to analyze the association between dietary diversity and overweight,obesity.Methods A representative sample of 674 rural residents was selected by a multistage sampling method from Jilin Province in 2012 June to July.A validated semi-quantitative food-frequency questionnaire was used to assess the usual food intake.The height and body weight were measured and the body mass index (BMI) was calculated.Logistic regression analysis was applied to calculate the risk of overweight and obesity for different DDS,after adjusted for mixed factors.Results 62.4% people in rural scored ≥6 while 11.8% people in  rural scored ≤3. The detection  rate of obesity of the  rural adults  in Jilin Province was higher than the mean level in China . For rural adults with moderate and adequate diversity score,the risk of overweight and obesity was  0.946 and 0.816 times the risk of overweight and obesity of the rural adults with pool diversity score.
Conclusion Diet diversity of the  rural adults  in Jilin Province is low.The risk of overweight and obesity is high;the risk of obesity is decreased with the increasing of diet diversity level.

Objective To establish the real-time fluorescence quantitative PCR method for the detection of bifidobacteria in human fecal samples,and to provide a
n effective means for measuring intestinal bacteria.Methods Total DNA of bacteria was extracted from 60 cases of children's fecal samples.Three primers of bif
idobacteria based on the 16S ribosomal RNA (16SrRNA) which possessed specialities of bacteria as amplified region were designed.The part of amplified 16SrRNA gene sequences was used as standard production.The serial dilution of standard was analyzed to build an absolute quantitative standard curve with SYBR Green Ⅰ dye method,and the bifidobacterium contents in sixty human fecal samples were calculated.The sensitivity of the reaction was calculated by detecting the lowest detectable standard which determined the sensitivity of the reaction.The PCR products’ melting curve was used to evaluate the specificity.The coefficient of variation (CV) of different batches of standard  with the same concentration was used to evaluate the stability of reaction.
Results The length of PCR product fragment which was used to build the standard curve was about 613 bp,the sequencing result was consist with the goals,and the standard sample of bifidobacteria was successfully established in real-time fluorescence quantitative PCR.The standard curve showed a good linear relationship with R2=0.999.The minimum detection value was 1.48×102 copies per reaction.The melting curve of real-time fluorescence quantitative PCR was a single peak.The test samples were batched and then examined by fluorescence quantitative PCR. The CV of standards’ Ct values which calculated from the  standard (1.48×103－1.48×107copies/ μL) were 2.94%,3.39%,3.54%,3.08%, and 3.34%,respectively.The contents of bifidobacteria in
fecal from 60 children was 7.77±0.86（copies?g-1 wet fecal）transformed by logarithmic.Conclusion The established real-time fluorescence quantitative PCR method has high sensitivity,strong specificity and good repeatability,which is suitable for detection of human fecal bifidobacteria content.