To investigate the effect of ionizing radiation on the expression levels of hyaluronic acid(HA) protein and hyaluronidase(HAase1) mRNA in human fibroblasts and to discuss the role of HA protein and HAase1 mRNA in the pathogenic mechanism of radiation-induced brachial plexus injury.Methods The human fibroblasts were divided into   0.5,1.0,2.0,4.0 and 6.0 Gy X-rays radiation groups and sham irradiation group (0 Gy). The morphological changes of the fiber cells after irradiated by different doses of X-rays were observed.The expression levels of HA protein and  HAase1 mRNA at 24  and 48 h after irradiated by different doses of 0,0.5,1.0,2.0,4.0 and 6.0 Gy X-rays were detected by ELISA and RT-PCR methods.Results The cells appeared vacuolization，and the nuclear chromatic began to get together after 2.0 Gy exposure;the nuclear saged and  the chromatin fractured after 4.0 Gy exposure;the nuclear saged,chromatin fractured and the part of cell plasma was dissolved after 6.0 Gy exposure.Compared with 0 Gy group,the expressions of HA protein were  increased significantly at 24 h after X-rays irradiation with the doses of 0.5,1.0,2.0,4.0 and 6.0 Gy(P<0.05) and the expression of HA protein had no  significant change  at 48 h after irradiation,but the expressions of HA protein in 4.0 and 6.0 Gy groups were increased significantly  compared with 0 Gy group (P<0.05). The levels of HAase1 mRNA were markedly increased at 24 h  after irradiated by different doses of X-rays (P<0.05).Compared with 0 Gy group,the expressions of HAase1 mRNA were increased significantly at 48 h after 1.0，4.0 and 6.0 Gy exposure compared with 0 Gy group(P<0.05).Conclusion The X-rays irradiation can induce  the increase of the expression of HA protein and the level of HAase1 mRNA in human fibroblasts ,and it may influence the occurrence and development of radiation-induced brachial plexus injury.

o construct human secreted TRAIL(hsTRAIL) recombinant vetor pEgr1-hsTRAIL mediated by Egr1,and to explore the inhibitory effect on the growth  of  lung adenocarcinoma A549 cells.Methods   The  hsTRAIL vetor mediated by Egr1 was constructed by gene recombination techinique,the A549 cells were transfected with the plasmid after identification by PCR,restrictive enzyme digestion and sequencing,and irradiated by 6 Gy X-rays.There were control group,pEgr1-hsTRAIL group,6 Gy  X-rays group and pEgr1-hsTRAIL + 6 Gy  X-rays group in the experiment.The expression of hsTRAIL　in A549 cells was detected by ELISA method,the cell proliferation was detected by MTT assay,the cycle changes of cell cycle were detected by flow cytometry and the  apoptosis was measured by TUNEL method.Results The hsTRAIL recombinant vector pEgr1-hsTRAIL mediated by Egr1 was constructed successfully.The cells were irradiated by 6 Gy X-rays after transfected with plasmid.The  hsTRAIL protein expressions in control,6 Gy and pEgr1-hsTRAIL groups didn’t change significantly with the time prolongation, but the expression in pEgr1-hsTRAIL+6 Gy group was increased significantly with the time prolongation (P<0.05 or P<0.01),and reached to peak value at 8 h.There was no significant difference of A549 cell proliferation ability between control group and pEgr1-hsTRAIL group,but the proliferation abilities in 6 Gy and pEgr1-hsTRAIL+6 Gy groups were decreased significantly compared with control group,especially in pEgr1-hsTRAIL+6 Gy group (P<0.05 or P<0.01).Compared with control group,the percentages of A549 cells  at different phases in  pEgr1-hsTRAIL group didn’t change significantly,but the percentages of A549 cells at  G0/G1 phase  in 6 Gy and pEgr1-hsTRAIL+ 6 Gy groups were  increased significantly  (P<0.05),the percentages of A549 cells at G2/M phase were decreased significantly  (P<0.05),the percentages of A549 cells at S phase didn’t change significantly.The percentages of A549 cells at different phases in 6 Gy group were basically consistent with those in pEgr1-hsTRAIL+6 Gy group.Compared with control group,the  apoptotic percentage of A549 cells in  pEgr1-hsTRAIL group had no significant change,but they were  increased significantly in 6 Gy and pEgr1-hsTRAIL+6 Gy groups compared with control group (P<0.01),especially in pEgr1-hsTRAIL+6 Gy group.Conclusion The hsTRAIL recombinant vector pEgr1-hsTRAIL mediated by Egr1 is successfully constructed,the vector can enhance the  inhibitory effects and  effect of inducing apoptosis of radiation on the A549 cells,but it affects cell distribution little.

To construct the prokaryotic expression system of the  recombinant advantage antigen epitopes of Leptospira,and to gain the high purity expression products of the  recombinant advantage epitope fusion protein of Leptospira and to provide the basis for research on  the  rapid  method to detect Leptospira.Methods The plasmid pET-rLP was constructed by gene recombinant method.The fusion protein with His-tag was efficiently expressed in the form of inclusion body after IPTG induction.The inclusion body was washed by urea,dissolved and purified by Ni2+ affinity chromatography,and it was identified by SDS-PAGE,Western blotting and ELISA methods.Results There was a specific protein band,which identified the high expression of the recombinant advantage epitope fusion protein of Leptospira by SDS-PAGE analysis and Western blotting,at the molecular weight of 20 000.The ELISA results showed specific reactions with Leptospirosis positive sera,but no cross-reaction with the other positive sera sample(salmonella) using the expression protein.Conclusion The prokaryotic expression system of the  recombinant advantage antigen epitopes of Leptospira is constructed successfully,and the recombinant advantage epitope of  fusion protein of Leptospira with high purity  is gained successfully.

To detect the expression  of angiopoietin-1(Ang-1) in mouse ovary tissue  and to discuss the influence  of Ang-1 on mouse follicular development,ovulation and formation and degradation of corpus luteum.Methods The sexual maturation model,follicular development induced by equine chorionic gonadotropin(eCG) model,ovulation induced by human chorionic gonadotropin (hCG) model and corps luteum formation and regression model were set up.The expressions of Ang-1 in the mouse ovary tissue at  different time in different models were detected by RT-PCR.Results The Ang-1 mRNA expression levels were lower on the 10th and 20th day after birth,but it was  increased on the  25th day after birth compared with the 10th day group(P<0.01);then it was gradually  decreased.The Ang-1 mRNA expression was increased compared with 0 h group(P<0.01) and it reached the  peak level at 48 h after injection of eCG.At 3,5 and 9 h after injection of hCG,the Ang-1 mRNA expression was decreased;while at 0.5 and 7.0 h after injection of hCG,the Ang-1 mRNA expression levels were higher;there were significant differences between   0.5 h group and other groups(P<0.01).The Ang-1 mRNA  expressed on  the 1st day to the 15th day  after injection of hCG,and on the 5 th day to the 15th day  after injection of hCG,the Ang-1 expressions were increased gradually compared with 1 d group(P<0.05 or P<0.01);and on   the 15th  day after injection of hCG,the  Ang-1 expression level reached  the peak.Conclusion Ang-1 may plays a role during the process of mouse follicular development,ovulation and corpus luteum formation and regression.

To compare the various indexes of mouse models of acute lung injury (ALI) established by exposed and   non-exposed intratracheal instillations in order to confirm which method was more suitable. Methods Forty-five male mice were randomly divided into control group,non-exposed group and exposed group.There were fifteen mice in each group.The mice in both non-exposed and exposed groups were instilled with lipopolysaccaride(LPS) to establish ALI models by  non-exposed and exposed intratracheal instillations,respectively. The detection of  biochemical indexes  in bronchoalveolar lavage fluid (BALF),differential cell counting in BALF,lung wet/dry  weight (W/D) ratio,and  pathomorphological observation of lung tissue were performed 24 h after intratracheal instillation.Results The success rate in establishing the mouse ALI model  in exposed group (100%) was higher than that in non-exposed group (86.7%). The total protein concentration in BALF,the alkaline phosphatase(ALP) and lactate dehydrogenase(LDH) activities,the amount of neutrophils in BALF,and lung W/D  ratio were significantly increased in non-exposed and exposed groups compared with control group (P<0.05).The total protein concentration in BALF,the ALP and LDH activities,the amount of neutrophils in BALF and lung W/D ratio in exposed group were significantly higher  than those in non-exposed group (P<0.05).The pathological changes in non-exposed group were characterized by pulmonary interstitial edema,but the exudative pulmonary edema was shown in exposed group.Conclusion The exposed intratracheal instillation is proved to be more suitable to establish the mouse model of ALI.

To observe the influence of pregnant and lactational D-galactose exposure in lipid peroxidation,hearing function and morphology and  structures  of cochlear hair cells  in newborn rats,and to clarify the relation with  cochlear hair cell injury,hearing loss and increased lipid peroxidation.Methods 12 pregnant rats were randomly devided into control group (n=6) and D-galactose group (n= 6) from gestational day 6,the rats in control group were fed on standard rodent diet,and the  rats in  D-galactose group were fed on standard rodent diet supplemented with 30% D-galactose.On postnatal day 21,the blood levels of galactose,superoxide dismutase(SOD),glutathion(GSH),glutathione peroxidase(GSH-Px) and malondialdethyde(MDA) in newborn rats were measured. Then  the distortion product otoacoustic emission (DPOAE)  was examined.The temporal bones were then removed and the morphological changes were detected by silver nitrate staining.Results The levels of blood SOD,GSH-Px and GSH were lower in newborn rats after using D-galactose during pregnancy and lactation compared with control group (P<0.05),and the plasma MDA level was higher than that in  control group (P<0.05).In addition,the DPOAE threshold shifts at different frequencies were lower after using D-galactose during pregnancy and lactation compared with  control group (P<0.05).The structures of cochlea hair cells were impaired and the number of hair cells was decreased in D-galactose group.Conclusion D-galactose exposure may cause oxidative and cochlear function damage in newborn rats.

To investigate the antioxidant activities of icariin and icariside Ⅱ,and to clarify the possible antioxidant mechanism.Methods Using Vc and butylated hydroxytoluene (BHT)as  positive controls,the clearance rates  of icariin ,icariside Ⅱ and BHT on 1，1-diphenyl-2-picryl-hydrazyl radical (DPPH?),superoxide anion(O２－?) and hydroxyl free radical (OH?) were measured;the reduction rates of samples were detected by the system of potassium ferricyanide;the  capacities of samples were detected by NADH-NBT-PMS system; the inhibitory rates of lipid peroxidation were detected by thiobarbituric acid (TBA) method;the  total antioxidant activities were determined by bleaching of β-carotene suboleic acid system.Results The samples showed  concentration-dependent scavenging capacities of  DPPH?.The clearance abilities of icariside Ⅱ  were higher than those of  icariin (P<0.05).The clearance effects of  icariin and icariside Ⅱ were concentraion-dependent;compared with the same concentration of BHT,the O2－?  clearance rates of icariside Ⅱ and icariin were slightly lower, and the clearance rate of icariin was much lower than that of icariside Ⅱ.The OH? clearance rates of icariin and icariside Ⅱ  in the concentration of 0.1-0.5 g.L^-1 were (16.76±0.35)% -(40.56±1.46)% and (15.65±0.72)% -(28.51±0.91)%. When the concentration was 0.9 g.L^-1,the inhibitory rates of  lipid peroxidation of icariin,Vc and icariside Ⅱ were  (58.79±1.56)%,(75.05±2.12)% and (37.82±1.43)%.The inhibitory rates no longer changed with the dose increasing.As the sample concentration increasing,the reduction capacities of  icariin,Vc and icariside Ⅱ showed  concentration-dependent tendence.In 30-120 min the  total antioxidant activity of icariside Ⅱ was lower than those of icariin and BHT(P<0.05 or P<0.01).The antioxidant activities of icariin and icariside Ⅱ in vitro  were weaker than those of Vc and BHT.Conclusion Icariin and icariside Ⅱ shows obvious antioxidant activities in  scavenging of DPPH?,O2-? and OH? and inhibition of lipid peroxidation, the total antioxidant activity and the reduction activity.

To explore the influence of   human thermal transient receptor potential 1 (TRP1)  gene transfection in expression of P27 protein in corneal endothelial cells   in cats and the effect of termal TRP1 gene in  proliferation cycles of corneal endothelial cells and to provide theoretical foundation for the application of the gene in human corneal endothelial cells.Methods 20 healthy full-term kittens were randomly divided into experiment group and control group.The corneal endothelial cells were collected after anesthesia and cultured.The cells in experiment  group were transfected with  thermal TRP1 gene mediated by liposome.The cells in control group were treated with 5.0 μL PBS instead of the expression vector into medium.The mRNA  expression  of thermal TRP1 gene was detected by RT-PCR.The expression of P27  protein  in cat corneal endothelial cells was observed by Western blotting.The influence of target gene transfection in the activities of cell division and regeneration 96 h after culture was observed.Results The inflow of calcium ions in the cultured cells  was increased significantly after heating.The RT-PCR  results of thermal TRP1 gene transfection showed that the strip density in experiment group was higher than that  in control group (t= 2.21,P= 0.037).The expression of  P27 protein in experiment group was significantly higher than that in  control group (t=2.53,P= 0.021).The differences of mitotic index and the proportion of the  cells at G1 phase  between experiment group and control group were statistically significant (χ2=3.26,χ2=4.18,P<0.05).Conclusion The human thermal  TRP1  gene transfection in cat corneal endothelial cells can increase the expression of  P27 protein and inhibit the proliferation of corneal endothelial cells.

To study the descending inhibitory effect of the lateral reticular nucleus (LRN) treated with electrical stimulation on the cardiosomatic motor reflex (CMR) induced by intrapericardial capsaicin (IC) in rats and its mechanism.Methods  22 male SD rats were randomly divided into electrical stimulation group(n=7),electrical stimulation plus 3 g?L-1 yohimbine group(n=5),electrical stimulation plus 5 g.L^-1 yohimbine  group(n=5) and electrical stimulation plus vehicle group(n=5).The  electromyogram (EMG)  changes  of dorsal spinotrapezius muscle of CMR induced by  IC of rats were observed following intervention with treatment factors in various groups.Results Compared with control group,EMG could be inhibited by electrical stimulation of 10,20 and 30  μA in  LRN,the inhibitory effect was increased with the increasing of  the electrical stimulation intensity accompanied or not accompanied with changes of blood pressure;the EMG reduced to 51.41%,21.11% and 1.43%  of the control(P<0.001);Compared with electrical stimulation(10  μA) group,intrathecal injection of adrenergic α2 receptor antagonist yohimbine (3 and 5 g.L^-1) reversed the inhibitory effects of LRN stimulation on CMR,the  EMG responses increased from 41.34% to 72.40% and 78.73%,respectively (P<0.01).Conclusion Electrical stimulation of LRN can produce descending inhibition on CMR and  α2-adrenoceptors at spinal cord level may be involved in the inhibition.

To isolate，cultivate and identify the hepatocellular carcinoma associated fibroblasts (hCAF) and to investigate their possible role in the occurrence and development of hepatocellular carcinoma.Methods The primary hCAF were isolated and purified by enzyme digestion method. The expressions of the specific proteins associated with hCAF were examined by Western blotting method. The changes of cell cycles of Hep3B cells after hCAF supernatant treatment were evaluated by flow cytometry. The effect of hCAF on hepatocellular carcinoma cells  was observed though building a xenograft tumor model in nude mice. Results The hCAF were successfully isolated and confirmed and highly specificly  expressed α-smooth muscle actin (α-SMA)  detected  by Western blotting method. The proliferation capacity of Hep3B was increased significantly after treated with hCAF conditioned media. The proliferation capacities of Hep3B were 126.0%,125.0% and 120.5% of those treated with complete medium for 48 h(P<0.05).The number of carcinoma cells and the percentages of carcinoma cells in S phase were higher than those treated with complete medium for  48 h(P<0.05).The tumor volume in Hep3B cells and hCAF co-injection group ［(1.34±0.52) cm2］ was significantly larger than that in Hep3B cells inoculation  group ［(0.51±0.09)cm2］ in nude mice(P<0.05). Conclusion hCAF could promote the proliferation of hepatocellular carcinoma cell line Hep3B, and  hCAF might play an important role in the progression of hepatocellular carcinoma.

To observe the influence  of  novel liver X receptor (LXR) agonist ATI-829 on the blood glucose level,body weight and dietary consumption of type 2 diabetes mellitus KKAy mice and  the incidence of type 1 diabetic NOD mice, and to provide evidence for clinical application.Methods Fifteen 12-week male type 2 diabetes mellitus KKAy mice were randomly divided into vehicle control group,pioglitazone treatment group,and ATI-829 treatment group.The mice were treated for 2 weeks, and the  blood glucose levels,body weights,and the dietary consumptions of mice were measured every week.Forty 6-week female type 1 diabetes mellitus NOD mice were divided into vehicle control group,classic LXR agonist T1317 treatment group,and ATI-829 treatment group.The mice were treated for 8 weeks, and the cumulative incidence was  measured every two weeks from 20 to 34 week.Results In type 2 diabetes mellitus mice,after 1 week treatment,the glucose level,body wieght and the dietry consumption of mice in various groups didn’t change significantly compared with those before treatment.After 2 weeks treatment, the blood glucose level of mice in pioglitazone treatment group was decreased from (30.11±3.94) mmol.L^-1 to (14.00±2.78) mmol.L^-1,and the body weight was  increased from (36.96±1.17) g to (41.14±0.99) g,there were significant differences compared with vehicle control group(P<0.05).The blood glucose level of mice  in ATI-829 treatment group was decreased from(33.28±1.89) mmol.L^-1 to (15.89±1.06) mmol.L^-1,there was significant difference compared with vehicle control group(P<0.05);and the body weight was  decreased from (37.82±0.96) g to (37.11±0.26) g,there was no significant difference compared with vehicle control group(P>0.05).The dietary consumption didn’t change significantly in various groups before and after treatment.In type 1 diabetic NOD mice,ATI-829 was able to totally prevent the disease occurrence and the disease incidence in T1317 treatment group rose to 90%,which was 50%  in vehicle control group，there were  significant differences between T1317 group,ATI-829 treatment  group and    vehicle control group(P<0.05).Conclusion The novel   liver X receptor  agonist ATI-829 can decrease the blood glucose levels of type 2 diabetes mellitus mice and avoid body weight gain.Meanwhile,ATI-829 can also decrease the incidence of type 1 diabetes mellitus in mice.

Objective
To explore the effect of ginsenoside Rb1 on hippocampal neuron injury after treated by Aβ-amyloid protein in rats and provide theoretical basis for its  clinic application.Methods The cultured hippocampal neurons from newborn SD rats were divided into normal control group,hippocampal neurons treated with Aβ-amyloid protein group (injury control group),low dose(10 mg/L) Rb1 group,middle dose(50 mg/L) Rb1 group, and high dose(100 mg/L) Rb1 group.Then the  survival rates of neurons were detected by MTT colorimetry and the apoptosis of neurons were detected by TUNEL staining，and the caspase-3 activities of neurons were detected by immunohistochemistry method.Results The MTT results showed that the survial rates  of hippocampal neurons were decreased in injury control  group,10 mg/LRb1 group,50 mg/LRb1 group and 100 mg/L Rb1 group  compared with normal control group(P<0.05).Compared with injury control group,the survial rates  in 50 mg/L Rb1 group and 100 mg/L Rb1 group were increased(P<0.05).The TUNEL results  showed that compared with normal control group,the apoptosis of neurons  in  injury control group,10 mg/L  Rb1 group,50 mg?L-1 Rb1 group and 100 mg?L-1 Rb1 group were increased(P<0.05).The aproptotic rate  of neurons in 100 mg/L Rb1 group was higher than that in  injury control group (P<0.05).The expression of caspase-3  in    100 mg/L Rb1 group was lower than that in injury control  group (P<0.05).Conclusion Ginsenosides Rb1 can protect hippocampal neurons against Aβ-amyloid protein injury,which is achieved through the regulation of apoptosis.

Objective
To study the effect of peroxisome proliferators-activated receptor-γ(PPAR-γ) ligands rosiglitazone(RSG) on focal cerebral ischemia injury   in type 2 diabetic(T2DM) rats from the endoplasmic reticulum stress（ERS） perspectives and to clarify its mechanism.Methods 72 male SD rats were fed with high amounts of sugars and fats for 4 weeks,then the low -dose STZ (25.0 mg/kg) was injected into caudal vein to make T2DM rat models, the  model rats were divided into four groups randomly:sham operation group(n=18),control group(n=18),RSG group(n=18) and RSG+GW9662 group(n=18).The MCAO models were made after the rats in each group were pretreated by drugs.The surgical procedure in sham operation group was the same as the above,but no thread inserted.The rats were executed  24 h after cerebral ischemia,the neuroethology evaluations for  the rats in each group   were made,and the  pathomorphology of brain tissue in rats  was  observed by HE staining,the expressions of glucose-regulated protein 78 （GRP78） and C/EBP homologous protein （CHOP）were detected by immunohistochemical and Western blotting,and the expressions of GRP78 mRNA and CHOP mRNA were detected by RT-PCR.Results  The nerve function defect after cerebral ischemia improved obviously in RSG group compared with control group(P<0.01);there were no statistically significant differences between RSG+GW9662 group and control group(P>0.05).The GRP78 expression in RSG group was higher than that in control group(P<0.05),however,the CHOP expression in RSG group was lower than that in control group (P<0.05)；RSG+GW9662 group showed opposite trend compared with RSG group.Conclusion PPAR-γ agonist RSG has the protective effect on focal cerebral ischemia injury  of T2DM rats and the mechanism may be related with up-regulating  the   GRP78 expression and  decreasing  the CHOP expression.

Objective To establish liver  injury rat models with lipopolysaccharide (LPS) and investigate the protective effect of erythropoietin (EPO) on liver injury and its possible mechanism,and to provid  evidence for the treatment of liver injury induced by LPS. Methods 40 adult Wistar rats were randomly divided into blank control group(n=10),EPO control group(n=10),LPS group(n=10) and LPS + EPO group(n=10). The  liver injury rat models were established by injecting 10 mg/kg LPS into the tail vein of  rats in LPS and LPS+EPO groups,while the rats in  control group received the same amount of saline. 30 min later,the rats in LPS + EPO group and EPO control group were given rhEPO (5 000 U/kg) via the tail vein injection,while the rats in the other  two groups were given saline.5 rats were killed in each group after 6 and 24 h,and the levels of serum enzymes including aspartate aminotransferase (AST) and alanine transarninase (ALT) were evaluated by biochemical analysis and the tumor necrosis factor-α (TNF-α) was determined by immunoradiometric assay. The other rats were killed at 24 h,the liver tissue sections were underwent histological examination by hematoxylin and eosin (HE) staining under light microscope. The ultrastructure changes of liver tissues were assessed by transmission electron microscope (TEM).The expressions of AKT,P-AKT and NF -κB were determined by Western blotting. Results Compared with control group,the levels of ALT,AST,and TNF-α in LPS and LPS + EPO groups were elevated(P<0.05);the levels in LPS group were increased significantly compared with LPS+EPO group (P<0.05). Compared with control group,the expressions of AKT,NF-κB in LPS group were enhanced; compared with  LPS group,the expressions of P-AKT and NF-κB in LPS+EPO group were decreased. The light microscope results showed the inflammatory  cells presented  infiltration and swelling or necrosis of liver cells in the liver tissue in LPS group,and the inflammatory cell infiltration and  hepatocyte swelling were also visible in LPS+EPO group,but much slightly. The electron microscope results  showed that the  hepatocyte mitochondrial presented swelling,disappearance of microvilli,vacuolization of liver cells in LPS group,and the similar injury was visible in LPS+EPOgroup,but much slightly. Conclusion EPO can protect the LPS-induced liver injury by reducing inflammatory and tissue degeneration which may be related with phosphatidylinositol 3-kinase (PI3K)/AKT/NF-κB signal pathway .

Objective
To  investigate the effect of mesenchymal stem cells(MSCs) modified by GATA4- pEGFP gene on heart function in myocardial infarction rats and to provide basis for further research on its mechanism.Methods The MSCs were isolated and cultured in vitro.GATA4-pEGFP plasmid was constructed and transfected into MSCs by liposome.40 SD rats were operated to prepare myocardial infarction models and the heart function indexes of the models were evaluated 10 d after the operation.The model rats were randomly divided into MSCs group (n=20) and DMEM group (n=20).The MSCs modified by GATA4-pEGFP gene was transplanted into the area of myocardial infarction of rats 10 d after infarction in MSCs group. The equal volume of  serum-free  DMEM was injected into the myocardial infarction area of rats at the same time in DMEM group.The  left ventricular ejection fraction (LVEF) and fractional shortening (FS) 4 weeks after injecting were detected.Results The isolated   MSCs in vitro  adhered to the plate and they presented fusiform or spindle shape.2 weeks after transfection with  recombinant gene plasmid GATA4-pEGFP,the green fluorescence protein  expressed in  MSCs.4 weeks after  transplantation,the LEVF value in MSCs group(85%±7%) was  increased significantly compared with DMEM group(47%±4%) (P<0.01),and the FS value in MSCs group  (52%±6%) was also increased significantly compared with  DMEM group  (35%±8%)(P<0.01).Conclusion The heart function of myocardial infarction rat can be improved by transplanting MSCs modified by GATA4 gene.

Objective
 To constructed an eukaryotic expression vector pcDNA3.1(+)-AKR1B10 and  observe its expression in breast cancer MCF-7 cells,and to provide experimental basis for  study on the relationship between AKR1B10 gene and breast cancer.Methods The  breast cancer  MCF-7 cells were transfected transiently with  the constructed eukaryotic expression vector pcDNA3.1(+)-AKR1B10. The expressions of AKR1B10 gene in the untransfected MCF-7 cells,the MCF-7 cells transfected with pcDNA3.1(+)-AKR1B10 and the MCF-7 cells transfected with pcDNA3.1(+) (control vector) were detected by RT-PCR and Western blotting methods.Results The eukaryotic expression vector pcDNA3.1(+)-AKR1B10 was successfully constructed.The RT-PCR results showed that if the AKR1B10 gene expression in the untransfected  MCF-7 cells was regarded as 1, the expression values  were 1.79 in the cells  transfected with pcDNA3.1(+)-AKR1B10 and  0.98 in  the cells tranfected with pcDNA3.1(+). The expression of AKR1B10 gene in the cells  transfected with pcDNA3.1(+)-AKR1B10 was significantly increased compared with the cells   transfected with pcDNA3.1(+) and the untransfected cells (P<0.05).The Western blotting results showed that the expression of AKR1B10 gene in the cells transfected with pcDNA3.1(+)-AKR1B10  was significantly increased compared with the cells transfected with pcDNA3.1(+)  and the untransfected cells.Conclusion The eukaryotic expression vector pcDNA3.1-AKR1B10 is successfully constructed.The AKR1B10 gene highly expresses in the MCF-7 cells transfected with the expression vectror.

Objective
 To construct the myocardial infarction models in rats with kidney-yang deficiency and study the differences in myocardial morphology,myocardial enzymology and hemorheology between the myocardial infarction models with kidney-yang deficiency and the simple myocardial infarction models in rats and to provide evidence for evaluation of pharmacodynamics of medicine for curing chest and heart pain.Methods  60 Wistar rats were randomly divided into control group,kidney-yang deficiency models group,myocardial infarction sham operation group,myocardial infarction models group and myocardial infarction models with kidney-yang deficiency group,and there were 12 rats in each group.The myocardial infarction models under the status of the kidney-yang deficiency in rats were set up to determine the parameters of myocardial infarct size (MIS) and the activities of serum aspartate aminotransferase (AST),creatine phosphokinase (CK) and lactate dehydrogenase (LDH),at the same time,the platelet agglutinate rate (PAR),platelet aggregation(PAG),erythrocyte sedimentation rate(ESR),hematocrit(HCT) and the length,the dry weight,the wet weight of the thromb in vitro were tested,the parameters of thrombelastogram were also be detected.Results There were no obvious differences in MIS,the activities of CK,AST and LDH,and PAR,PAG,ESR and HCT between the myocardial infarction models with kidney-yang deficiency in rats and the simple myocardial infarction models（P>0.05）;the dry weight of the thromb and the length of thromb in vitro of the myocardial infarction models with kidney-yang deficiency in rats were heavier than those of the simple myocardial infarction models and the kidney-yang deficiency models in rats,but there were no significant differences（P>0.05）.The increasing extent of the values of r,k and the reducing extent of the value of ma in the myocardial infarction models with kidney-yang deficiency in rats were higher than those in the simple myocardial infarction models,but there were no significant differences between them（P>0.05）.Conclusion The obvious differences are not showed in the parameters of the indexes of MIS,the myocardium enzyme in the serum,HCT and ESR,the indexes of the function of the blood plate,the weight of the thromb in vitro and the thrombelastogram.

Objective To study the effect of fibroblast growth factor-21 (FGF-21)on the lipid metabolism of  nonalcoholic fatty liver disease（NAFLD）cell model  induced in vitro and its potential mechanism.Methods The concentrations of medical fat emulsion (MFE) and FGF-21 were selected by MTT assay.And the NAFLD cells were divided into  control group,model group,leaving fat emulsion group and FGF-21 therapy group.Oil red O staining was used to observe lipid droplet under light microscope.The intracellular  triacylglycerol (TG) levels  in each group were observed by hol-automatic biochemistry.The expressions of carnitine palmitovl transferase-1 (CPT-1),peroxisome proliferator-activated receptor-gamma (PPARγ) and sterol regulatory element binding protein-1c (SREBP-1c) were determined by immunocytochemistry.Results The 0.1% medical fat emulsion (MFE) was chosen by  MTT assay as the best concentration for the induction of NAFLD cells in vitro.The intracellular TG level of NAFLD  cells was increased significantly compared with normal liver cells HL7702 (P＜0.01),and there was a large quantity of orange-red lipid droplet in cytoplasm of NAFLD cells;meanwhile,the expression of CPT-1 protein got lower,and the expressions of  PPARγ  and SREBP-1c proteins got higher.MTT assay showed the best management concentration of FGF-21 was 0.5 mg/L for setting up  NAFLD-effect model,the intracellular TG level in  FGF-21 group was decreased significantly compared with model group(P＜0.01);the expression level of CPT-1 protein was increased (P＜0.01),and the expressions of  PPARγ and SREBP-1c proteins were decreased in  FGF-21 group.Conclusion  FGF-21 is helpful to relieve accumnlation of TG in NAFLD cells,and it decrease the expressions of PPARγ and SREBP-1c protein and increase the expression of CPT-1 protein in HL7702 cells.

Objective
 To study the effect of 20(s)-proto-panaxdiol ( PPD) on apoptosis of human prostate cancer PC3 cells cultivated in vitro,and to clarify the mechanism of its anticancer action.Methods The PC3 cells cultivated in vitro were divided into control, and 20,30,40  μmol/LPPD groups.The morphological changes of PC3 cells were observed under optical microscope after treated with PPD for 48 h.The proliferation inhibition of PC3 cells was detected by MTT method.The apoptosis of PC3 cells was detected by AO/EB staining and the  expressions of Bax,Bcl-2 and γH2Ax proteins were analyzed by Western blotting.Results After treated with different doses of PPD,the PC3 cells were round,recovery,and broken.AO/EB staining found that the cells presented yellow-green.The inhibitory rates of proliferation of PC3 cells in  various  groups were increased compared with control group(P<0.05).The  expression of Bcl-2 protein was down-regulated(P<0.05), the expression of Bax protein didn’t change significantly,  and the expression  of γH2Ax protein was up-regulated(P<0.05).Conclusion PPD may induce the apoptosis of PC3 cells,and its mechanism may be related to the down-regulation of Bcl-2 protein and the up-regulation of γH2Ax protein expression.

Objective
To investigate the inhibitory effect of juglone on the proliferation of leukemic cells and to clarify the mechanism of anti-leukemia of juglone.Methods The leukemic cell lines K562,Jurkat,U937,U266 and NB4 were cultured in vitro and divided into 4 groups as follows: zero group,containing medium,MTT and triple lysate;blank control group,containing a variety of human leukemia cells,drug dissolution media,culture medium,MTT and triple lysate;experiment groups,respectively using different concentrations(final concentration: 0.25,0.50,1.00,2.00,4.00,6.00,8.00,16.00 mg/L)of juglone disposal of various cell lines.The inhibitory effects of juglone of proliferation of leukemia cells were detected by MTT method.The apoptosis of K562 cells after treated with different concentrations of juglone was detected by flow cytometry (FCM).Results Juglone had  strong inhibitory effects on the K562,Jurkat,U937,U266 and NB4 cell lines.Their IC50 (48 h) were 3.87,1.13，4.48,1.87 and 4.67 mg/L,respectively,and the inhibitory effects on Jurkat and U266 cells (P<0.05) were most obvious.The IC50 values  of juglone on other three leukemia cell lines (48 h) were less than 40 mg/L,and the inhibitory effects of juglone on these five cell lines presented dose-and time-dependent manner.FCM analysis showed that juglone could induce the apoptosis of K562 cells;the apoptotic rates of K562 cells were (4.54±0.6) %,(11.5 4±0.6) %,(28.7±0.3) %,(45.0±1.2) % and (76.1±1.4) %,respectively, after treated with  1,2,4,8 and 16 mg?L-1 juglone for 6 h.Conclusion Juglone can inhibit a variety of leukemia cell lines in vitro,and the mechanism may be related to  promoting the apoptosis of K562 cells.

Objective
To study the effect of growth hormone relasing peptide(GHRP) on the IL-1β level in myocardium tissue of chronic heart failure(CHF) rats and to discuss the relative mechanism.Methods 30 healthy male Wistar rats were randomly divided into normal group(n=10),model group (n=10) and treatment group (n=10).The CHF models were set up by injecting adriamycin,the hemodynamic changes were observed and the pathological changes of the myocardium tissue of rats were observed by HE staining;the morphological changes of myocardial cells were detected by electron microscope;the changes of the forms and the mean density of immunological positive cells of IL-1β  were detected by immunohistochemistry method;the changes of the contents of IL-1β in myocardial tissue of rats were detected by ELISA method.Results Compared with normal group,the heart rates,the arterial pressures and the maximum change rates of left ventricle pressure in model group were  decreased significantly(P<0.05);the indexes mentioned above in treatment group were a little lower than those in normal group,but there were no significant difference(P>0.05).The myocardial cells in model group arranged irregularly and presented inflammatory invasion;the morphological changes of myocardial cells in treatment group presented little compared with normal group.The electron density of intercalated disc in model group was decreased,and the mitochondrial appeared swelling and vacuolar degeneration in many regions;whereas the mitochondrial in treatment group kept original structure and the swelling and vacuolar degeneration appeared in a few regions.The immune-positive cells in model group tended to be hypertrophy,and the cells showed a phenomenon of aggregation,but the positive cells in treatment group were reduced significantly and showed a phenomenon of scatteration;the mean density of expression of IL-1β in model group was obviously higher than those in normal group and treatment group(P<0.05),the mean density of expression of IL-1β in treatment group was a little higher than that in normal group,but there was no significant difference(P>0.05).The content of IL-1β in myocardial tissue in model group was higher than those in normal group and treatment group obviously(P<0.05);the contents of IL-1β in treatment group were a little higher than those in normal group (P>0.05).Conclusion GHRP can decrease
the content of IL-1β in myocardium tissue and  improve the myocardial cells remodeling  in  CHF rtas.

Objective
To investigate the hepatotoxicity and DNA damage induced by thioglycolic acid (TGA)-capped cadmium telluride (CdTe) quantum dots(QDs) in human hepatic cells HL-7702,and to provide experimental evidence for the toxicological research and safety evaluation.Methods The HL-7702 cells were divided into control group and CdTe QDs-treated groups with different concentrations of CdTe QDs(0,6.25,12.50,25.00 and 50.00 mg/L, respectively).After  treated with CdTe QDs,the cell proliferation was measured by MTT assay,the DNA damage by single cell gel electrophoresis (SCGE),the cell cycle process by propidium iodide (PI)-flow cytometry (FCM) and the  apoptosis by acridine orange/ethidium bromide (AO/EB) assay and Annexin Ⅴ- fluorescein isothiocyanate (FITC)/PI-FCM.Results The MTT results showed that CdTe QDs inhibited the proliferation of HL-7702 cells in a dose-dependent and time-dependent manner.The results acquired by SCGE and FCM with PI staining showed that after CdTe QDs exposure for 24 h,as exposure dose increased,the degree of DNA damage raised, and the percentage of HL-7702 cells in G0/G1 phase was decreased while they were significantly increased in S and G2/M phases (P<0.05).In AO/EB assay,the apoptotic cells were observed under fluorescence microscope;In Annexin Ⅴ-FITC/PI-FCM,the apoptotic rates in CdTe QDs groups were significantly increased compared with  control group (P<0.05). Conclusion CdTe QDs could influence the cell viability,and induce DNA damage,S and G2/M phases arrest and apoptosis in HL-7702 cells.

Objective To construct  overexpression vector of miR-210 and to identify the miR-210 target gene  HBVSP2 by using a dual fluorescent protein repoter assay system.Methods The primary sequence of miR-210 was cloned to form a new plasmid named pcDNA3.1(+)/pri-miR-210.A sequence of  HBVSP2 was inserted into the plasmid which expressed green fluorescent protein (EGFP) pcDNA3/EGFP.The plasmid pcDNA3/EGFP-HBVSP2 and miR-210ASO or pcDNA3.1(+)/pri-miR-210 and the plasmid expressed red fluorescent protein (RFP) pDsRed2 -N1  were cotransfected into HEK 293 cells and  HepG2 2215 cells.The fluorescence value  of the extracted protein was detected by fluorescence spectrophotometer respectively.Results After pcDNA3/miR-210 and the plasmid of pcDNA3/EGFP-HBVSP2 being cotransfected,the indensity of EGFP/RFP   was significantly lower than that in   pcDNA3+pcDNA3/EGFP-HBVSP2 group(P<0.05).The EGFP/RFP was lower in PCDNA3/pri-miR-210+pcDNA3/EGFP-HBVSP2 group compared with  pcDNA3+pcDNA3/EGFP-HBVSP2 group(P<0.05).After miR-210 ASO and the plasmid  pcDNA3/EGFP-HBVSP2 being cotransfected,the indensity of EGFP/RFP was significantly higher than that in  pcDNA3/EGFP-HBVSP2+LacZ group(P<0.05).The EGFP/RFP was lower in LacZ+pcDNA3/EGFP-HBVSP2 group compared with  LacZ+pcDNA3/EGFP group(P<0.05).Conclusion The miR-210 overexpression plasmid pcDNA3.1(+)/pri-miR-210 and pcDNA3/EGFP-HBVSP2 contained miR-210 target condition are successfully constructed,and HBVSP2 may be a direct target gene of miR-210.

Abstract:Objective To explore the adjuvant effect of stimulating epitope  of mycobacterium tuberculosis heat shock protein 70(mtHSP70) on  fusion gene vaccine   of hepatocellular carcinoma associated antigens 661(HCA661), and to provide basis for improving immune effect of gene vaccine. Methods Eighteen BALB/c mice were randomly divided into three groups:pCI-neo group(n=6),pCI-HCA661-HSP70C group(n=6) and pCI-HCA661-HSP70E group(n=6).The  BALB/c mice were immunized intramuscularly with empty plasmid pCI-neo,recombinant plasmid pCI-HCA661-mtHSP70C and pCI-HCA661-mtHSP70E.The  changes of T cell subsets were analyzed by FCM. The antigen-stimulated production of IFNγ in the supernatants of  splenocytes in vitro and the specific  IgG antibody against HCA661 were detect by ELISA. The specific CTL cytotoxicity against tumor cells was used to detect the inhibitory effect on tumor cells. Results The percentagse of CD3+,CD4+T lymphocytes and the ratio of CD4+/CD8+ were significantly increased in  pCI-HCA661-mtHSP70C group and pCI-HCA661-mtHSP70E group compared with pCI-neo group（P<0.05）. The percentages of CD3+,CD4+ T lymphocytes and the ratio of CD4+/CD8+ were  significantly increased in pCI-HCA661-mtHSP70E group compared with pCI-HCA661-mtHSP70C group(P<0.05). The concentration of IFNγ in pCI-HCA661-mtHSP70E group was higher than that in pCI-HCA661-mtHSP70C group in supernatants from cultural splenocytes under HCA661 peptide stimulation（P<0.05）. The IFNγ levels in  pCI-HCA661-mtHSP70C group and pCI-HCA661-mtHSP70E group were higher than that in pCI-neo group(P<0.05).The levels of specific antibodies induced by fusion gene in pCI-HCA661-mtHSP70E group were increased significantly compared with pCI-HCA661-mtHSP70C group（P<0.05）.The anti-tumor effect of CTL was enhanced strongly in pCI-HCA661-mtHSP70E group. Conclusion Stimulating epitope of mtHSP70 can induce a strong specific immune response to HCA661 fusion gene vaccine in mice by its adjuvant effect.

Abstract:Objective  To examine the regulation of neuropeptide Y (NPY)  on sweet taste in the central nucleus of amygdala(CeA) and to explore its possible mechanism.Methods 58 male SD  rats were chosen,and 10  rats of them were divided into drinking saccharin solution group(n=5) and drinking distilled water group(n=5);after 2 h,the rat brain was obtained.The expressions of NPY and c-Fos in CeA were detected by immunohistochemical staining method.The other 48  rats were implanted cannula in CeA.The postoperative 24 rats were randomly divided into 4 groups,and the rats in 2 groups were injected with NPY and the rats in the other 2 groups were injected with  saline in CeA,and the intake of saccharin solution and distilled water were detected 2 h later.The other postoperative 24 rats were randomly divided into 4 groups,and the rats in 2 groups were intraperitoneally  injected with naloxone and the rats in the other 2 groups were intraperitoneally injected with saline,after 2 min,the rats were injected with NPY or saline in CeA,and the intake of saccharin solution were detected 2 h later.Results The NPY and c-Fos expressions in CeA were significantly increased after drinking saccharin solution (P<0.05).The intake of saccharin solution was significantly increased after injecting NPY in CeA(P<0.05);the pre-treatment of naloxone blocked the orexigenic effect of NPY(P<0.05).Conclusion NPY in CeA may play an important role in the regulation of sweet taste of saccharin solution,and the mechanism may be related to the opioid system.

Abstract:Objective To induce bone mesenchymal stem cells (MSCs) of rabbits to differentiate directionally into chondrocytes in vitro and to observe the morphology of cells and detect the function,and to discuss the necessary differentiation condition in vitro.Methods The MSCs and chondrocytes were isolated from rabbits and cultured in vitro.The supernatant of chondrocytes in the first three passages  were collected and used to induce transformation of MSCs for 14 d.Three experimental groups were set up: negative control group,experimental group and positive control group.After 14 d of induction,the  morphology of the cells in each group was observed，and the specific  proteins were observed by toluidine blue staining,and  the alkaline phosphatase (ALP) activities were detected，and the collegen Ⅱ(Col Ⅱ) mRNA expressions were detected by RT-PCR.Results 14 d after  induction,compared with negative control group,the induced MSCs in experimental group changed into polygonal shape from spindle shape and there was positive  appearance of toluidine blue staining,the ALP activity and Col Ⅱ mRNA expression were  increased significantly compared with the other two groups(P<0.01).In negative control group, there was negative  appearance  of toluidine blue staining and Col Ⅱ mRNA expression.In  positive control group, there was  positive  appearance  of   toluidine blue staining and Col Ⅱ mRNA expression.Conclusion The supernatant of chondrocytes from rabbits can promote the  MSCs differentiate into chondrocytes in vitro.

Abstract:Objective To clone human truncated apoptosis inducing factor (AIF) cDNA sequence,and to construct early growth response 1 (Egr1)-mediated recombinant expression vector pcDNA3.1-Egr1-AIFΔ1-480 (pEgr1-AIFΔ1-480),and to observe its regularity induced by radiation in human breast cancer   MCF-7 cells.Methods The total mRNA extracted from human leukemia jurkat cells used  as  template,and the human AIFΔ1-480 was acquired by RT-PCR,and it was linked to pMD18T vector for sequencing.Egr1 fragment was digested from pMD19T-Egr1 by restrictive enzyme,and the  Egr1-mediated expression plasmid pEgr1-AIFΔ1-480 was constructed by gene recombination.There were control group,pcDNA3.1 group,pAIFΔ1-480 group and pEgr1-AIFΔ1-480 group in the experiment. After the  plasmids in various groups were transfected into human breast cancer MCF-7 cells,the AIF and AIFΔ1-480 protein expression time-effect (0,2,4,12,24 and 48 h after 2.0 Gy irradiation) and dose-effect (24 h after 0,0.2,0.5,1.0,2.0 and 5.0 Gy irradiation) regularity were measured by Western blotting method.Results The sequencing results showed that the AIFΔ1-480 acquired by RT-PCR was consistent with the sequence expected，the pEgr-AIFΔ1-480 was confirmed  by PCR and restrictive enzyme digestion.After 0-48 h the MCF-7 cells were irradiated by 2.0 Gy,and the AIF protein expressed in the cells in each group,and it increased significantly from 4 h and the AIF expressions in 4,12,24 and 48 h groups were  higher than that in 0 h group(P<0.05),and it reached to maximum value at 48 h.But the AIFΔ1-480 protein expressed  in the  cells in pAIFΔ1-480 and pEgr1-AIFΔ1-480 groups from 2 h (P<0.05),and it reached to peak value at 24 h.The AIFΔ1-480 expressions in pEgr1-AIFΔ1-480 group were higher than those in pAIFΔ1-480 group at 24 and 48 h (P<0.05).After the  MCF-7 cells were irradiated by 0-5 Gy for 24 h,the AIF protein expressed in the cells in each group,but the AIFΔ1-480 protein expressed merely in the cells in pAIFΔ1-480 and pEgr1-AIFΔ1-480 groups.The AIF and AIFΔ1-480 expressions were increased with the dose increasing,the AIF expressions irradiated with 0.2，0.5，1.0 and 5.0 Gy were  higher than that with 0 Gy irradiation.It reached to maximum value by 5.0 Gy irradiation and the AIFΔ1-480  expression  in pEgr1-AIFΔ1-480 group was  higher than that in  pAIFΔ1-480   group.Conclusion The human truncated AIF expression recombinant vetor pEgr1-AIFΔ1-480 is successfully constructed,and the  AIF and AIFΔ1-480 protein expressions increased with time prolongation and dose increasing after irradiation in MCF-7 cells.

Abstract:Objective To detect the expression of Twist1 protein in gastric carcinoma tissues,and to explore the clinicopathological significance of Twist1 protein in the carcinogenesis and progression of gastric carcinoma.Methods The expressions of Twist1 protein were detected by immunohistochemistry in 199 cases of gastric carcinomas,25 cases of high-grade intraepithelial neoplasia,24 cases of low-grade intraepithelial neoplasia,46 cases of intestinal metaplasia,17 cases of chronic atrophic gastritis and 23 cases of normal gastric mucosa.The relationships between the expression of Twist1 protein and clinicopathological characteristics of gastric carcinoma were analyzed.Results The expression of Twist1 protein in gastric carcinoma tissue was higher than those in intraepithelial neoplasia,chronic atrophic gastritis and normal gastric mucosa tissues,and there were  significant differences（P<0.001）.Meanwhile,the expression of Twist1 protein in tumor size ≥4 cm group was higher than that in tumor size <4 cm group（P<0.01），and it was higher in the patients with  lymph node metastasis than that in patients without metastasis（P<0.01）,and it was higher in the died patients  than that in lived patients （P<0.01）.However,the 　expression of Twist1 protein was not correlated with the type of gastric carcinoma,patient’s age,tumor location,T stage,tumor differentiation，clinical stage and  histological type of gastric carcinoma.Conclusion The expression of Twist1 in gastric carcinoma tissue is high,and it is related to tumor size and metastasis and prognosis,which may be an important factor in occurence of gastric carcinoma.

Abstract:Objective To evaluate the association between  brain lesion location and occurrence of poststroke depression  patients  (PSD) with acute stroke from Chinese people with Meta-analysis,and to provide evidence to explore mechanism of PSD.Methods Pubmed,CNKI,VIP and Wanfang databases were searched by computer to find literatures focused on associations between  lesion location and PSD from January 2000 to November 2011.Those met criterion were included in the study and  evaluated.The data in the articles was  extracted at the same time.ReviewManager 5.1 software was performed to analyze the data. Fix effect model and random effect model were used to calculate combined effects.Publish bias was evaluated and the sensitivity analysis was performed.Results Twenty-two articles were included in the study finally.In all,3 545 subjects included,which contained 1 493 PSD patients and 2 052 non-PSD persons.Combined OR and its 95% confidence interval which were used to evaluate associations between PSD and different   lesion location were as follows: left hemisphere 1.60(1.39-1.83) (P<0.001),front of brain 2.76(1.60-4.75) (P<0.001),frontal lobe 2.11(1.00-4.45) (P=0.05),and cortex 2.22(0.74-6.62) (P=0.15).Among acute stroke patients,those who suffered with their left hemisphere had 1.6 times higher risk to be PSD of those with right hemisphere injure;the patients who suffered with their front of brain had 2.76 times higher risk to be  PSD of those with back of brain injure.Conclusion The  lesion places  which located in left hemisphere and front of brain are the risk factors for PSD.

Abstract:Objective  To investigate the effects of overexpression of DHRS7 gene and short hairpinRNA(shRNA) recombinant plasmid on cell cycles of the human breast cancer MCF-7 cells and the relationship between DHRS7 and breast cancer invasion,and to clarify the effect of DHRS7 on the development of breast cancer.Methods  The cDNA fragment encoding human DHRS7 was obtained from mRNA of breast cancer cells MCF-7 by RT-PCR.Then it was cloned into vector pcDNA3.1(+) plasmid to conduct DHRS7-shRNA-pGenesil.There  were four groups in the experiment:  pcDNA3.1-DHRS7 group,negative control group(pcDNA3.1 group),blank control group and DHRS7-shRNA-pGenesil group.Then the plasmids were transfected into MCF-7 cells using Fugene HD transfection agent,and the expressions of DHRS7 were determined by RT-PCR and Western blotting.The cell cycles were analyzed by flow cytometry.The expression of DHRS7 protein in carcinoma in stiu and infiltrating carcinoma were detected by immunohistochemical staining.Results The recombinant plasmid pcDNA3.1-DHRS7 was constructed correctly.RT-PCR analysis and Western blotting showed that the expression of DHRS7 protien in pcDNA-DHRS7 group was higher than that in blank control group（P<0.05）,and the expression of DHRS7 protien in DHRS7-shRNA-pGenesil group was lower than that in blank control group（P<0.05）.Flow cytometry showed the overexpression of DHRS7 increased the cells in S phase （P<0.05） and the low expression of DHRS7 increased the cells in G2 period（P<0.05）.Immunohistochemical staining showed that DHRS7 protein expression was higher in the  breast cancer  in situ,and the positive rate was 100%(37/37)；the DHRS7 protein expression  was lower in the breast infiltrating carcinoma tissues,and the positive rate was 18.9% (7/37),there was significant difference between them(P<0.01).Conclusion DHRS7 gene may be involved in the regulation of cell cycle in MCF-7 cells,which can inhibit cell proliferation and the expression missing of DHRS7 protein may promote invasion of  breast cancer.

Abstract:Objective To discuss the effect of cyclooxegenase-2（COX-2） selective suppressor celecoxib on the expressions of vascular endothelial growth factor(VEGF) mRNA and matrix metalloproteinases-9(MMP-9) mRNA in human medullary thyroid TT cells,and to approach the anticancer mechanism of celecoxib.Methods  The TT cells of human medullary thyroid carcinoma were divided into control group and different concertrations of celecoxib groups,and the cells in celecoxib groups were treated with different concentrations  of celecoxib（20,40 and 80  μmol?L-1）,the cells in control group were treated with  F12K culture fluid.The expressions of VEGF mRNA and MMP-9 mRNA in each group were detected by RT-PCR 72 h after treatment. Results the RT-PCR results showed that the expressions of VEGF mRNA and MMP-9 mRNA in  TT cells were decreased  after  treated with  20,40 and 80  μmol?L-1 celecoxib. There was  significance difference of the VEGF/GAPDH and MMP-9/GAPDH of TT cells between 40,80  μmol?L-1 celecoxib groups and   control group（P<0.05）.Conclusion The secretion of VEGF mRNA and MMP-9 mRNA is decreased obviously when the concentration  of celecoxib is above 40  μmol?L-1,which may inhibit the growth of tumor.

Abstract:Objective To observe the changes  of the  levels of serum interleukin-10（IL-10）,plasma endothelin(ET-1) and immunoglobulin E (IgE) in the children with or without  atopic asthma and to discuss their  clinical significances.Methods 60  asthma children conducted the Allergen skin prick tests and serum IgE measurement to determine atopic status at the same time,and they were divided into atopic asthma group(n=32) and non-atopic asthma group(n=28) according to the results. 30  normal healthy children were selected as control group.The expressions of ET-1 and IgE in the asthma children during attack period and remission phase and the children in control group  were detected by radioimmunoassay and the  levels of serum IL-10 were detected by the double antibody sandwich ELISA.Results The  level of serum IL-10 in the asthma children in the acute attack period was lower than those in control group(P<0.01), and the level of ET-1 was higher obviously than that in control group(P<0.05);the levels of IgE in the asthma children in the acute attack period and remission phase in asthma groups were obviously higher than that in control group(P<0.01). The levels of ET-1 and IgE of the atopic asthma children in acute attack period were higher than those of the atopic asthma children in remission phase,and the level of IL-10 was lower(P<0.01).The levels of serum  IL-10 in atopic group of the acute attack period was lower than that in non-atopic group(P<0.05),and the levels of the ET-1 and IgE of the patients in  the acute attack period and remission phase in atopic group were higher than those in non-atopic group(P<0.05).The relationships between IL-10 level  and ET-1 and IgE showed  obviously negative correlations(r=－0.592，r=－0.894，P<0.05),and the relationship between ET-1 and IgE showed  obviously positive correlation(r=0.623，P<0.05).Conclusion The  levels of serum IL-10 and ET-1 perhaps take  part in the pathologic and physiological process of the children’s asthma and the changes of  the  levels of IL-10,ET-1 and IgE in the children with  atopic asthma  are more obvious.

Abstract:Objective To investigate the thermostability of GTPase activity of cell division protein FtsZ in Porphyromonas gingivalis（Pg) and to discuss the biochemical properties of FtsZ, and to provide experimental basis for curing periodontal disease.Methods Malachite green assay was used to measure the  the GTPase activities of recombinant Wt PgFtsZ,ZΔC01(missing 73 residues from the C-terminus of PgFtsZ),ZΔC02(missing 128 residues from the C-terminus of PgFtsZ),ZΔC03(missing 177 residues from the C-terminus of PgFtsZ),and ZΔC04(missing 233 residues from the C-terminus of PgFtsZ).The GTPase activities of different kinds of proteins were detected  after the protein dealed with unboiling and boiling for 5 and 10 min.The values of GTPase activities were detected at A650nm.Results The GTPase activities of ZΔC04  boiled for 5 and 10 min were higher than those of unboiled(P<0.05).The GTPase activities of WtPgFtsZ,ZΔC01,ZΔC02,and ZΔC03  boiled for 5 min were higher than those of unboiled(P<0.05).But the GTPase activities of WtPgFtsZ,ZΔC01,ZΔC02,and ZΔC03  boiled for 10 min were similar to those of unboiled.Conclusion Pg FtsZ  is a kind of  protein with thermostability,and ZΔC04 missing 233 residues from the C-terminus has higher thermostability.

Abstract:Objective To study the T lymphoctye subsets and Th1/Th2 type cytokines levels in peripheral blood of  patients with hepatitis B infection at different stages and healthy people and its clinical significance and to provide experimental basis for diagnosis and  therapy of  the diseases. Methods The percentages of CD3+,CD4+ and CD8+ T lymphocytes among total lymphocytes in the peripheral blood of patients with HBV infection at different stages including 14 cases of moderate chronic hepatitis B(CHB), 7 cases of severe CHB,8 cases of severe hepatitis B and 5 cases of acute hepatitis B and 20 healthy controls were detected by flow cytometry. The levels of IL-2,IL-4 and IFN-γ in serum were detected by ELISA method.Results Compared with  control group,the levels of  CD3+,CD4+T lymphocytes and the levels of IL-2 and IFN-γ in the patients in acute hepatitis B group were increased significantly（P<0.05）；the levels of  CD3+ and CD8+T lymphocytes and the level of  IFN-γ in patients in severe hepatitis B group were higher （P<0.05）； the levels of IL-4 and IFN-γ in the patients in moderate and severe CHB groups were increased （P<0.05）.Conclusion Th1 cytokines might play a role in the development of hepatitis B and the increasing of Th2 cytokine may be related to the chronic process  in the patients with HBV infection.

Objective To study  the   inhibitory effect of celecoxib on proliferation of    adiramycin(ADM)-resistant  breast cancer cells MCF-7/ADM  and to discuss the anti-tumor effect of  celecoxib.Methods The MCF-7/ADM cells treated with different concentrations of ADM (0.05,0.10,0.20,0.40,0.80,1.60  mg/L) were used as  control groups;the MCF-7/ADM cells treated with cell-free complete medium were used as negative control group and   the cells treated with ADM and  10,20  μmol?L-1  celecoxib were  used as experiment group.The inhibitory rates of cells in various groups were detected by CCK-8 after 24,48 and 72 h.The MCF-7/ADM cells treated with  0.05  mg?L-1 ADM and  different concentrations of celecoxib (10 and 20  μmol/L) were collected 3 h  after treatment,the concentrations of ADM were detected by flow cytometry in various groups.Results The growth of cells were inhibited  24,48 and 72 h after treated  with different concentrations of ADM alone or combined with celecoxib (10 and 20  μmol/L). The inhibitory rates of cells in experiment group were higher than those in contronl group(P<0.05);the inhibitory rates in 20  μmol/Lcelecoxib experiment group were higher than those in 10  μmol/L celecoxib experiment group at  24,48 and 72 h （P<0.05）.The cells in control groups changed little but the cells in experiment group changed a lot,which showed a celecoxib dose-dependent manner.Compared with control groups,the concentrations of ADM in the cells  in experiment group were increased(P<0.05).The concentration of ADM in the cells in 20 μmol/L celecoxib experiment was higher than that in 10 μmol/L celecoxib eperiment group(P<0.05).Conclusion The selective COX-2 inhibitor celecoxib combined with ADM can reverse the ADM resistance  of   breast cancer cell lines.

Objective
To assess the efficacy and adverse reactions of imatinib in the treatment of patients with chronic myeloid leukemia (CML),and to provide the basis for choosing the best treatment program. Methods 239 patients diagnosed as CML were divided into three groups,55 patients in CML chronic phase (CP)  CML  group were treated with imatinib,30 patients in the accelerated phase (AP) and blastic phase (BP)  group were treated with imatinib and 154 patients in CML  CP chemotherapy and/or interferon  group were treated with chemotherapy and/or interferon.The clinical symptoms after treatment,such as the  complete hematologic remission (CHR),the complete cytogenetic remission (CCR),the major molecular remission (MMR) and the side effects were analyzed.The plasma concentration,the chromosome and the mutation of the imatinib-resistant patients were deteced.Results 46 patients in  CP CML group  were in the early CP (the patients were treated within 1 year after  diagnosis,CP <1 year).100% of the patients in this group achieved CHR in 3 months,and 83% of the patients achieved CCR in 12 months,and 30% of the patients achieved MMR in 18 months.There were 9 patients in the late CP (the patients were treated more than 1 year after the diagnosis,CP> 1 year),the CHR rate was 88%,and the CCR rate was 30%,and the MMR rate was 22%. In AP and BL CML group,there were 12 AP CML patients,50% of the patients in this group achieved CHR,and 25% of the patients achieved CCR,and 17% of the patients achieved MMR;there were 18 BP CML patients,22% of the patients achieved CHR,and 16.7% achieved CCR,and no patient achieved MMR.In  CP CML chemotherapy and/or interferon group,the CHR rate was 47.4%,and both the CCR and the MMR rates were  0%.The CHR,CCR and MMR of the patients in CP CML  group were higher than those in CP CML  chemotherapy and/or interferon group(P<0.05). 14 patients in  imatinib groups were imatinib-resistant.The drug concentrations of 2 imatinib-resistant patients were tested to be declined.5 imatinib-resistant patients had complex karyotypes(including 2 CP patients,2 AP patients and 1 BP patients).The gene mutations were tested in 4 imatinib-resistant patients.Conclusion Compared with CP CML chemotherapy and/or interferon group,the patients in imatinib groups could achieve a higher hematologic,cytogenetic and molecular remission rate.The patients in  CP CML group treated with imatinb achieve a higher remission rate than AP and BP CML group.The remission rate of the patients in  early CP group is higher than that in late CP group.

Objective To evaluate the short-term efficacy and safety of intensity modulated radiation therapy(IMRT) plus concomitant and adjuvant temozolomide (TMZ) in patients with  malignant gliomas and to provide basis for selection of clinical therapy program．Methods Forty-six postoperative patients with malignant gliomas confirmed by pathology were randomly divided into combined therapy group(n=22,IMRT+TMZ) and radiotherapy group(n=24,IMRT alone).All the patients were followed up for 12-31 months,and the median follow-up time was 19 months.The median progression-free survival,1-year survival rate and the incidence of adverse reactions  were observed.Results All the patients were carried out according to the plan.The median progression-free survival was 10 months in combined therapy group and 7.6 months in radiotherapy group;the 1-year survival rates were 86.36% and 58.33%（P<0.05）,respectively.The main adverse reactions in two  groups were myelosuppression and gastrointestinal reactions of gradeⅠ-Ⅱ,there was no significant difference between two groups（P>0.05）,and  the patients could tolerate well.Conclusion The combination of IMRT and TMZ can achieve much better short-term efficacy than IMRT alone,and the adverse reactions are little,which is a safe and effective method for postoperative maglignant glioma.

Objective To detect the root curvature and diameter in human maxillary anterior teeth by cone-beam computed tomography (CBCT) and to provide some anatomical parameters related to post-core design. Methods A total of 129 human maxillary anterior teeth were selected and analyzed.The three-dimensional images of these teeth were obtained by multiplanar reconstructions (MPR) technique of CBCT,and the root curvature and diameter were observed and measured by dedicated software.  Results The mean labio-lingual root curvature degree  of maxillary central incisors was significantly smaller than those  of maxillary lateral incisors and canines (χ²=6.592，P=0.037),while the  labio-lingual root curvature  radius  was significantly larger than those of other  groups （χ²=8.504，P=0.014）. There were significant differences in the root length distribution of the mesio-distal curved part  between the three different maxillary anterior teeth groups（χ²=13.910，P=0.008）. The mean diameter measured labiolingually was significantly different from that measured mesiodistally  in various groups（P=0.000）. Conclusion  There are differences in root morphology of 129 human maxillary anterior teeth,and the root curvatures and diameters of human maxillary anterior teeth  present differently in CBCT.
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Objective To analyze the relationships between quality of life (QOL) and socio-demographic and clinical characteristics of patients with pulmonary heart disease(CPHD),and  to discuss the clinical significance of the QOL score changes.Methods The survey was conducted in 141 CPDH patients who were in the acute exacerbation.Using the QLICD-CPHD（V2.0）which was a QOL scale for CPHD patients to detect the data.The data was assessed at the time of patients’ admission and dischange to the hospital.The relationships between QOL and socio-demographic and clinical characteristics were analyzed by  statistical methods.Results QOL was negatively correlated with the  alanine aminotransferase,the aspartate amino transferase,the urea,the blood platelet count,the PaCO2 and the body temperature;and QOL was positively correlated with the total protein,the kalium,the leukocyte number,the neutrophil absolute value,the red blood cell count,the PO2 and the lung functional parameters.Lung function was the influence factor of the QOL.The influence factors of the body function included FVC,FEV1, EV1/FVC and PEF;the influence factors of the psychological function included FEV1,EV1/FVC and PEF;the influence factors of the social function included PEF and FVC;the influence factors of the specific module included FVC,FEV1,EV1/FVC and PEF;the influence factors of the total scale scores included FVC,FEV1,PEF and  EV1/FVC.The  95% confidence interval of  QOL was analyzed by combinating with  the lung functional classification,then  the clinical significances  were revealed when the minimal changes of scores were 5.25 in  physical function,3.27 in  social function,2.91 in  psychological function,7.62 in  specific module and 10.85 in the overall.Conclusion  The  change of QOL score can reflect the clinical degree and condition of CPHD patients.

Objective
 To explore the effects of 8-week aerobic interval training on cardiac function and physical fitness in obese teenagers and to provide evidence for formulating special exercise prescription for obse teenagers.Methods Forty male obese teenagers (BMI ≥ 25) were randomly divided into control group (n=20) and exercise group (n=20).People in exercise group conducted a 8-week (twice per week,50-60 min per session) aerobic interval training and people in control group maintained their routine lifestyle.The heart rate (RHR),systolic blood pressure(SBP),diastolic blood pressure(DBP),maximal oxygen uptake (VO2max),percentage of body fat and cardiac function parameters including left ventricular end-systolic volume (LVESV),left ventricular end-diastolic volume (LVEDV),stroke volume (SV),cardiac output (CO),fractional shortening (FS) and left ventricular ejection fraction (LVEF) were determined by echocardiography  before and after the intervention in two groups.Results There were no significantly differences of indicators between two groups before test.After test,the percentage of body fat,RHR and SBP in exercise group were decreased (P<0.05 or P<0.01);VO2max  was increased (P<0.01);the  LVEDV,SV,LVEF and FS were elevated (P<0.05 or P<0.01) and CO had no significant difference compared with before test;but the indexes in control group showed no significant changes.The percentage of body fat,RHR and SBP in exercise group after test  were lower than those in control group(P<0.05 or P<0.01);VO2max, LVEDV,SV,LVEF and FS  were higher than those in control group(P<0.05 or P<0.01).Conclusion Aerobic interval training may improve cardiac function and physical fitness in obese teenagers.

Objective
 To study the effect of different cultivate time,ultrasonic time and ultrasonic power on the concentration,type and quantity of the  intracellular protein from Candida albicans and Fusarium solina and to provide the theoretical basis for establishing a reliable and stable extraction method for intracellular protein from fungus.Methods Candida albicans and Fusarium solina were cultured.The different parameters included the culture time(36,48,and 72 h),ultrasonic time(20 s,3 min,5 min,and 10 min) and ultrasonic power(332.5 W and 475.0 W).The concentrations,species and quantities of protein under different factors were detected by the protein concentration detection and SDS-PAGE electrophoresis analysis.Results Both strains got the most quantity of protein when cultivated for 36 h.The most quantity and species of Candida albicans were obtained when the ultrasonic power was 332.5 W and ultrasonic time was 3 min,but as to Fusarium solina,the corresponding parameters were 332.5 W and 10 min, respectively.Conclusion The intracellular protein is extracted by compositely using ultrasonic method and glass bead transformation method.The optimal extraction method of intracellular protein from fungus is developed from the aspects of culture time,ultrasonic power and ultrasonic working time of strains.

Objective
To isolate  the flavonoid glycosides from Scorzonera austriaca Willd and identify its structure and to provide experimental evidences for medicinal values of flavonoid glycosides from Scorzonera austriaca Willd. Methods The herbs of Scorzonera austriaca Willd was extracted with 70% ethanol,the extractives were purified with macroporous resin,silica gel,ODS column chromatography and HPLC and the structure of componuds were identified. Results Three compounds were isolated,and their structures were identifited through spectroscopic analysis and physico-chemical constants.They were 5,7,4′-trihydroxy-flavone-8-C-β-D-glucopyranoside,5,7,4′-trihydroxy-flavone-6-C-α-L-arabinosyl-8-C-β-D-glucopyranoside and 5,7,3′,4′-tetrahydroxy-flavone-6-C-α-L-arabinosyl-8-C-β-D-glucopyranoside.Conclusion Three compounds are all regarded as flavonoid lycosides isolated from Scorzonera austriaca Willd for the first time.
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Objective
To develop the method of high performance liquid chromatography(HPLC) for determination of curcumin and curcumin derivative A in rat plasma and study their  pharmacokinetics in rats, and to provide basis for  clinical application of curcumin.Methods The curcumin and the curcumin derivative A were lavaged to the rats,and the concentrations of curcumin and curcumin derivative A in rat blood at different time points were determined by HPLC.The curcumin and curcumin derivative A were separated well on a Hypersil ODS2 C18 （5  μm×4.6 mm　×200 mm）under 30℃.The mobile phase of curcumin was a mixture of acetonitrile-water containing 1% acetic acid (45∶55) at a flow rate of 1 mL?min-1.The detective wavelength was 420 nm;the mobile phase of curcumin derivative A was a mixture of acetonitrile-water containing 1% acetic acid (70∶30) at a flow rate of 1 mL?min-1.The detective wavelength was 361 nm.Results The calibration curve was liner at the range of 0.25-100.00 mg?mL-1.The regression equation of curcumin was  Y=105.90X－22.70，r=0.999 6；the regression equation of curcumin derivative A was Y=71.20X+24.62，r=0.999 4.The RSD of intra-day precision and inter-day precision was less than 4%.The average recovery was more than 96%.The plasma concentration-time curves of curcumin and curcumin derivative A in rats  fit to one compartment model respectively.The clearance rate,the area under temperature and t1/2 of curcumin derivative A were 3.57,4.09 and 2.79 times respectively than those of curcumin.Conclusion The method of HPLC for the determination of curcumin and curcumin derivative A in plasma is accurate,simple and reliable,which provides the basis for the improvement of stablility and the extending of the half-time of curcumin.