Abstract:Objective To investigate the effect of X-irradiation on cell cycle progression of Jurkat and EL-4 cell lines. Methods The time- and dose-effects of cell cycle progression in Jurkat and EL-4 lymphoma cell lines after X-irradiation with different doses (1.0,2.0 and 4.0 Gy) were analyzed by flow cytometry (FCM) following staining with propidium iodide (PI). Results In time-effect experiment,it was demonstrated that the percentages of Jurkat cells in G₀/G₁ and S phases were decreased significantly after 2.0 Gy X-irradiation（P<0.01）,whereas G₂+M phases cells were increased significantly at the same time compared with sham-irradiation（P<0.01）.It was also showed that the percentages of EL-4 cells in G₂+M and G₀/G₁ phases were increased（P<0.05,P<0.01）,and S phase cells were decreased significantly（P<0.01）. For dose-effect experiment,the results showed that the percentages of Jurkat cells in G₀/G₁ and S phases were decreased significantly 16 h after X-irradiation with the doses of 1.0,2.0 and 4.0 Gy（P<0.05,P<0.01）,and G₂+M phases cells were increased compared with sham-irradiation（P<0.01）. The percentage of EL-4 cells in G₀/G₁ phase was increased（P<0.01）,and S phase cells were decreased（P<0.01）.However,the percentage of EL-4 cells in G₂+M phases was increased significantly only after 4.0 Gy X-irradiation（P<0.01）. Conclusion The cell cycle progression of Jurkat and EL-4 lymphoma cell lines could be changed by X-irradiation with the doses of 1.0,2.0 and 4.0 Gy. X-irradiation（1.0,2.0 and 4.0 Gy ）could induce G₂ arrest in Jurkat cells,G₂ and G₁ arrest in EL-4 cells.

Abstract:Objective To study the effect of low dose cyclophosphamide on function of CD4⁺CD25⁺Treg of mice with Lewis lung cancer and investigate the relationship between CD4⁺CD25⁺Treg and the genesis and development of tumor.Methods The models were divided into three groups:CTX group,tumor group and control group.In CTX group, models were established by injection CTX (25 mg•kg^-1) and subcultivated Lewis lung cancer cells were injected subcutaneously to the right axilla of C57BL/6 mice 7 d later.The dynamic changes of tumor volume were observed.The changes of the number of CD4⁺CD25⁺Treg were detected by flow cytometer and the expression of Foxp3 mRNA in spleen was analyzed by semi-quantitative RT-PCR.The changes of T lymphocyte proliferation in spleen were detected by MTT test.The changes of T lymphocyte killing function in spleen were detected by LDH releasing test.Results Compared with tumor group,CTX treatment might delay the development of tumor in 19 d-mice with Lewis lung cancer.The number of CD4⁺CD25⁺ Treg in spleen of mice in CTX group was lower than that in tumor group (P<0.05).The expression of Foxp3 mRNA in spleen lymphocyte in CTX group was significantly lower than that in tumor group (P<0.05).The changes of T lymphocyte proliferation in spleen in CTX group were significantly higher than that in tumor group (P<0.05).The changes of T lymphocyte killing function in spleen in tumor group was not significantly lower than that in CTX group (P>0.05). Conclusion After CTX injection,the number of CD4⁺CD25⁺Treg and the expression of Foxp3 mRNA in spleen of mice with Lewis lung cancer decrease,the immunological function of mice with Lewis lung cancer increase.It can provid experiment evidence for the tumor immunotherapy aimed directly at CD4⁺CD25⁺Treg.

Abstract:Objective To study the changes of hematopoietic growth factors secreted by human mesenchymal stem cells from bone marrow(BM-MSC) pretreated with low dose radiation （LDR）.Methods The cultured P4 and P5 BM- MSCs were exposed to X rays atthe doses of 50 ，75 and 100 mGy (dose rate 12.5 mGy•min^-1).The changes of levels of stem cell factor (SCF), IL-6, macrophage colony-stimulating factor( M-CSF) secreted by BM-MSCs pretreated with LDR were determined by ELISA method. Results As compared with control group at the same time, the levels of SCF in experimental group had a tendency of increasing after 24 h and 48 h radiation,but only in 75 mGy group the SCF level was obviously increased (P<0.05). The levels of IL-6 in 50 and 75 mGy groups at 24 h and 48 h ,in 100 mGy group at 24 h were obviously increased compared with control group(P<0.05) .The levels of M-CSF in all the groups at 24 h, 48 h and 72 h except for the 50 mGy dose at 72 h were also increased(P<0.05),it increased markedly in 75 mGy dose group at 72 h.Conclusion LDR has hormesis effect on BM-MSCs .After LDR,the BM-MSCs grow faster and in a certain phase the expression levels of hematopoietic growth factors are increased.

Abstract:Objective To investigate the expression of ionic channels of the rat bone marrow mesenchymal stem cells（BMSCs）　and provide the basis for the change of ionic channels in the process of differentiation of stem cells.Methods The full marrow from Wistar rats was isolated by Percoll centrifugation and passaged repeatedly.The membrane currents of BMSCs were recorded with the whole-cell patch-clamp technique.The current amplitude of pathway, peal value and time course of activation and deactivation, selectivity of ions pathway，I-V curve were recorded.The currents were identified and analyzed by using various channel blockers．Results The cells began to stick in 24 h and arranged in whirlpool shape after passenger culture.They were positive for CD71, CD44, and negative for CD34, CD45 by flow cytometry, which improved that the cultured cells were BMSCs.The functional ion channels were characterized in cultured rat BMSCs with whole-cell patch clamp and reverse transcription polymerase chain reaction (RT-PCR) techniques.Three types of outward currents were found in BMSCs, including a delayed rectifier K⁺ current (IKDR)，in addition, tetrodotoxin-sensitive sodium current (INa.TTX) and nifedipine-sensitive L-type Ca²⁺ current (ICa.L) were detected in 24% and 25% BMSCs.Moreover, RT-PCR result revealed the molecular evidence of mRNA for the functional ionic currents, including SCN5A, Kv4.3 and CACNA1C.Conclusion The currents are consistent with the characteristics of excitable cell membrane.

Abstract:Objective To investigate the effect of ketamine on the synaptic long-term potentiation (LTP) in the CA1 area of rat hippocampal slices，and to elucidate the mechanisms underlying the effect of ketamine on memory.Methods Hippocampal slices (400 μm thick) were obtained from the brains of male Sprague-Dawley rats (2 months old) weighing 200-250 g that were decapitated.The slices were incubated in artificial cerebrospinal fluid (ACSF) at room temperature for at least 120 min before use.Forty-nine slices were randomly divided into 7 groups (n=7): control　 group, ketamine 1, 5, 10, 30, 50 and 100 μmol•L^-1 groups.All the slices in each group were perfused with ACSF, ketamine 1, 5, 10, 30, 50 or 100 μmol•L^-1, respectively.The slices in each group were performed to record evoked population spikes (PS) using extracellular microelectrode recording technique.Another forty-nine slices were randomly divided into 7 groups (n=7): LTP group, ketamine-LTP 1, 5, 10, 30, 50 and 100 μmol•L^-1 groups.All the slices in each group were perfused with ACSF, ketamine 1, 5, 10, 30, 50 or 100 μmol•L^-1, respectively.PSs were recorded for at least 30 min before LTP in each group.For LTP induction, high-frequency stimulation (HFS) conditioning pulses (100 Hz•s^-1) were applied to the Schaffer collateral-commissural pathway of hippocampus using a bipolar stimulating electrode.The changes in PS amplitude after HFS were analyzed in each group.Results The PS amplitude of the rat hippocampal slices in ketamine 1, 5, and 10 μmol•L^-1 groups had no significant difference compared with control group.The PS amplitude in ketamine 30, 50 and 100 μmol•L^-1 groups decreased compared with control group (P<0.01).The PS amplitude in group LTP after HFS was significantly increased by (52±12)% compared with pre-HFS, it indicated the successful induction of LTP.The amplitudes of the PS in ketamine-LTP 1 and 5 mol•L^-1 groups did not significantly change after HFS, when compared with LTP group.Compared with LTP group, the amplitudes of the PS in ketamine-LTP 10, 30, 50 and 100 μmol•L^-1 groups after HFS decreased significantly (P<0.01).Conclusion The inhibition of LTP induction in hippocampus of rats may contribute to ketamine-induced deficits in memory, and the underlying mechanism is involved in the inhibition of N-methyl-D-aspartate (NMDA) receptor in hippocampus.

Abstract:Objective To explore the protective effect of the polysaccharides of tricholoma matsutake(PTM) on hematopoietic function of X-rays irradiated mice.Methods Fifty ICR mice were divided into five groups randomly，10 each group．The mice in normal control group(NC) and radiation control group(IC) were gaster-poured by 0.9% sodium chloride solution．The mice in PTM groups were gaster-poured by three different doses PTM (400,800,1200 mg•kg^-1) for 7 d.The mice all groups except NC group were irradiated once with 2.0 Gy X-rays on the eighth day.The conony forming units-spleen (CFU-S),fibrolast colony forming unit (CFU-F),cell cycle of bone marrow cells and micronucleus rate of bone marrow polychromalic erythrocytes (PCE) were detected after radiation for 24 h.Results Compared with IC group,the number of CFU-S in PTM groups decreased significantly(P<0.05 or P<0.01)and the number of CFU-F increased markedly(P<0.05 or P<0.01);in high and moderate doses PTM the micronucleus rates of bone marrow PCE and the percentage of bone marrow cells at G₁ phase decreased signficantly( P<0.05 or P<0.01); the percentage of cells at S phase increased signficantly( P<0.05 or P<0.01).　Conclusion PTM has an obvious protective effect on hematopoietic function of X-rays irradiated mice.

Abstract:Objective To clone and express human basic fibroblast growth factor (hbFGF) and investigate its function.Methods The total RNA was isolated from human glioma cells and hbFGF was cloned by RT-PCR.After the PCR product was ligated with pMD18-T and sequenced,the pPICZα-hbFGF recombinant plasmid was constructed by subclone and transformed into Pichia pastoris X-33 via electroporation which was linearized with SacI.The fusion protein was verified and the transformant Pichia pastoris with high effective expression of hbFGF was screened by using PCR,SDS-PAGE and Western blotting.Results The obtained hbFGF gene was consistent with the hbFGF reported in GenBank (Accession no.NM002006).The hbFGF was expressed and secreted in X-33.SDS-PAGE and Western blotting analysis showed that the expressed protein in the culture supernatant induced by methanol was approximately 18 000 and could react specifically with anti-hbFGF antibody.Conclusion hbFGF gene is cloned successfully and expressed in Pichia pastoris.

Abstract:Objective To observe the leukogenic effect of tremella fuciformis spores polysaccharide (TSP) on leukopenia mice induced by cyclphohamide(CTX) and X-rays. Mice were randomly divided into six groups:normal control group (untreated normal animals); negative control group;positive control group;small,middle,large doses TSP groups.The mice of each group (except normal control group) were administered with CTX（100 mg•kg^-1）　 intraperitoneally or exposed to X-rays to result in leucopenia.In the normal and negative control groups,each animal was administered with saline; in TSP groups,mice were administered with TSP 25,50,100 mg•kg^-1 through caudal vein every day for 15 d; in positive control group,each animal was administered with matrine (200 mg•kg^-1) through caudal vein every day for 15 d.The numbers of peripheral leukocytes of all animals were counted before CTX treatment or exposure to X-rays and 3，6,9,12 and 15 d after treatment.The number of bone marrow karyotes of the mice was examined.Results Compared with normal control group,the leucocytes and the number of bone marrow karyotes of mice were remarkably decreased in the other groups after treated with CTX or exposure to X-rays (P<0.05),but leucocytes and the number of bone marrow karyotes were increased in TSP 50 and 100 mg•kg^-1 groups compared with negative control group (P<0.05).Conclusion TSP has the effect of the leukocytopoiesis promoting in the leukopenia mice induced by CTX and X-rays.

Abstract:Objective To establish E.coli strain expressing human IL-2 and MUC1 fusion protein to provide experimental fundation for a novel MUC1 cancer vaccine.Methods Human IL-2 gene fragments were obtained by PCR and they were inserted into recombinant vector pBS-SK-MUC1.After digested,human IL2-MUC1 gene fragments were cloned into prokaryotic expression vector pGEX-4T-1 and E.coli BL21 was transformed by constructing pGEX-IL2-MUC1.IL2-MUC1 fusion protein expression was induced by IPTG and analyzed by SDS-PAGE and Western blotting.Results Acquired gene fragments of human IL-2 (402 bp) and tandem IL2-MUC1 (852 bp)　 were identified by digestion and DNA sequencing. The recombinant vector of pGEX-IL2-MUC1 was constructed successfully.SDS-PAGE result proved that GST-IL2-MUC1 fusion protein with a relative molecular mass of about 64 000 was expressed.Western blotting result showed that the expressed products had specific reaction with both rabbit anti-human MUC1 polyclonal antibody and mouse anti-human IL-2 monoclonal antibody.Conclusion The engineering E.coli strain expressing human IL2-MUC1 fusion protein is successfully established.It is a basis to further study on novel cancer vaccine with MUC1 as target.

Abstract:Objective To study the effects of methyl mercury(MeHg) on oxidative damage, changes of cell cycle, apoptosis, and apoptosis-related protein expression in male mouse germ cells. Methods The cytotoxicity of MeHg on germ cells of male mice was measured by MTT assay. The content of malondiadehyde (MDA) and hydrogen peroxide (H₂O₂) and the activitiy of glutathione peroxidase(GSH-Px) in testicle germ cells were detected by TBA and KI colorimetric method and OPA fluorescence method. Apoptotic rate was measured by flow cytometry (FCM) and acridine orange/ethidium bromide(AO/EB) double fluorescent staining. The change of cell cycle was measured by FCM.Apoptosis-related protein expressions of Caspase-9 and CytC were detected by immunohistochemical method. Results When the cells were exposed to 0.1,1.0,10.0 and 100.0 μmol•L^-1 MeHg for 2, 4, 8, 24 and 48 h in vitro,the survival rates were decreased with the increasing of the dose of MeHg.The survival rates in groups of 1.0, 10.0, 100.0 μmol•L^-1 MeHg had significant difference compared with control group (P<0.05).After treatment with MeHg at the dose of 1/100 LD₅₀, 1/50 LD₅₀, 1/10 LD₅₀ to the mice, the results showed that with the increasing of MeHg, the contents of MDA and H₂O₂ in testis were increased.The contents of MDA in all the groups treated with MeHg were increased significantly compard with control group (P<0.05).The contents of H₂O₂ in 1/50 LD₅₀ and 1/10 LD₅₀ groups treated with MeHg were increased significantly compared with control group (P<0.01); with the increasing of MeHg,the activity of was GSH-Px decreased(P<0.05). The apoptotic rates in treated groups at the dose of 1/10 LD₅₀ were significantly higher than that in control group (P<0.05). The percentage of cells at G₂/M phase was increased with the increasing of MeHg, and the 1/50 LD₅₀ group had significant difference compared with control group (P<0.05).The expressions of Cyt C were increased in treated groups, and had significant difference compared with control group (P<0.01).The expressions of Caspase-9 were increased in treated groups, the groups of 1/50 and 1/10 LD₅₀ had significant differences compared with control group (P<0.01). Conclusion MeHg can induce cell oxidative damage, changes of cell cycle, apoptosis and changes of apoptosis-related protein expression in male mouse cells.MeHg has cytotoxic effect on male mouse germ cells.

Abstract:Objective To study the function of Bak in mitochondrial signaling pathway and interaction between Bax and Bak during apoptosis.Methods Bax/Bak double knock out (Bax^-/- and Bak^-/-)MEF cells from mouse embryo fibroblasts (MEF) and Hela cells were divided into four groups according the cell different genotypes(wild type,Bax^-/-,Bak^-/- and double knock out) and treated with different chemical reagents after co-transfection with Bax,Bak,Mito-Red and empty pEGFP vector.Apoptosis,mitochondrial morphology and cytochrome C release were detected with confocal microscope,immunofluoresence and Western blotting techniques.Results There were correlations between the percentage of Hela cell apoptosis and mitochondrial fission(%) as well as cytochrome C(%) (P<0.01).When Bak gene deletion,the apoptotic rate of MEF cells(17.9%) induced by chemical reagent was lower than that of wild type MEF cells(71.3%)(P<0.01); When gene double knock out MEF cells were transfected with GFP-Bak,mitochondrial fission incresed from 43.7% to 76.4%,the apopotic rate in pEGFP-Bak group was significantly higher than that in GFP-Bax group(P<0.05) and the same result was gotten when treated with different chemical reagents. Bcl-2 can not inhibit the apoptosis induced with Bak.Conclusion Bak can induce cell mitochondria fission and cytochrome C release and result in apoptosis.Bak can induce more cell apoptosis than Bax and the effect stands alone besides Bax action.

Abstract:Objective To investigate the degeneration of dopaminergic neurons induced by MPP⁺ in mouse mesencephalic dissociated culture and establish the cell model for parkinson’[KG-*3]s disease( PD) research in vitro.Methods OF1/SPF mouse embroys of 14 d were used to make mesencephalic dissociated culture,and the culture were divided into control and MPP⁺-treated groups.MPP⁺ with different final concentrations (0.1,1,10 and 15 μmol•L^-1)　 were added into the cultures on the 10th day in vitro,and incubated for 48 h.Then the dopaminergic neurons were identified by TH immunostaining.Hoechst 33342 was used to identify the apoptotic cells.Results The number of dopaminergic neurons was 875±23/well in control group,while there were 612 ±25,586±32,459±16 and 435±19 dopaminergic neurons per well in the 0.1,1,10 and 15 μmol•L^-1 MPP⁺-treated groups respectively,the numbers of dopaminergic neuron decreased significantly in MPP⁺ groups compared with control group（P<0.05）. On the 12th day in vitro,(5.45±0.29 )% cells in control group appeared as typical apoptotic appearance,a higher apoptotic rate was found in MPP⁺ group (26.97%±1.36% )(P<0.05).Conclusion All the concentrations of MPP⁺（0.1,１,10 and 15 μmol•L^-1） could induce the damage and dead of dopaminergic neurons. The degeneration and death are both dose- and time-dependent.Apoptosis may play an important role in the damage of dopaminergc neurons induced by MPP⁺.

Abstract:Objective To construct an eukaryotic expression vector pcDNA3.1-LIF by cloning mouse LIF genes and provide basis for establishment of transgenic animal models. Methods Total RNA was extracted from the tissue of the ICR mouse uterus .The full-lenth LIF gene of the mouse was amplified by RT-PCR and an eukaryotic expression vector pcDNA3.1-LIF with neomycin resistant was constructed by homologous recombination.Results The　 amplfied fragments was LIF cDNA about 612 bp, it had 100% homogeneity with mouse LIF cDNA sequence by nucleotide sequence analysis. An eukaryotic expression vector pcDNA3.1-LIF with neomycin resistant of the mouse was successfully constructed by the identification of enzyme digestion.Conclusion The mouse LIF gene is successfully cloned and an eukaryotic expression vector pcDNA3.1-LIF is successfully constructed.

Abstract:Objective To determine the effects of morphine on contents of glutamate (Glu) and glutamine (Gln) and transcripts of the related metabolic key enzymes in C6 glioma cells.Methods C6 glioma cells were cultivated and divided into 4 groups: control (C) group, morphine (M) group, morphine + naloxone (N+M) group, naloxone (N) group.Chromatometry was used to determine the contents of Glu and Gln in supernatant of cultured C6 glioma cells.RT-PCR was used to examine the relative mRNA contents of glutamine synthetase (GS) and glutamic acid dehydrogenase (GDH), which are the key enzymes of the Glu and Gln cycle.Results The extracellular Glu content was increased in M group compared with C group（P<0.01）, while it was decreased significantly in N + M group compared with M group (P<0.01); the extracellular Gln content in all three groups except C group showed a decreasing tendency (P>0.05).GS mRNA transcripts were decreased in M or N group compared with C group (P<0.01), while they were increased in N + M group compared with M group (P<0.01); GDH mRNA transcripts were increased in M group compared with C group (P<0.05), and were increased in N +M group compared with M group (P<0.01).Conclusion Morphine might change the extracellular concentrations of Glu and Gln by regulation gene expressions of GS and GDH, the key enzymes of the Glu and Gln cycle, which might be related with the effects of morphine dependence.

Abstract:Objective To explore the characterization of mitochondrial DNA and cytochrome b (Cytb) gene from Martes zibellina L..Methods The mtDNA were extracted from fresh heart and liver of Martes zibellina L. by alkaline lysis method respectively, and Cyt b gene segment was amplified by PCR technique and sequenced. Results The　 complete 16800 bp mtDNA was separated from both samples, and the Cytb gene with 310 bp segments was amplified by PCR and the sequence indicated that the homologous similarity was 99% with sable Martes zibellina L.(GenBank: AB026109.1) and it was 45% with other animal similarity.Conclusion There is complete mtDNA in tissues of Martes zibellina L. and Cytb partial gene from martes zibellina L.may be used as the genetic marker for identification of different species.

Abstract:Objective To observe the effect of Kudiezi Oral Solution (KDZOS) on total cholesterol(TC), lipoprotein cholesterol metabolism and antioxidative activity in experimental hyperlipoproteinemia rats.Methods 60　 Wistar rats were divided into 6 groups and each group had 10 rats.Control group was given normal food.The models of experimental hyperlipoproteinemia were established by giving lipid-riched feeds to Wistar rats for 15 d.The contents of TC,triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were measured and the atherogenic index(AI) was calculated.At the same time, the prostacycline (PGI₂) and thromboxane (TXA₂) levels in plasma, the malondialdehyde(MDA) contents and the superoxide dismutase (SOD) activities in serum and liver, and the viscosity of whole blood were also measured respectively in hyperlipidemia rats.In addition, the fat accumulation in liver was observed. Results In rats treated with KDZOS (with doses of 20 and 40 g•kg^-1 i.g for 30 d), the levels of TC, TG, LDL-C, TC/HDL-C, LDL-C/HDL-C, AI, TXA₂ ，MDA and the viscosity of whole blood were decreased significantly(P<0.05 or P<0.01), HDL-C, PGI₂, PGI₂/TXA₂ and SOD were increased significantly(P<0.05 or P<0.01), meanwhile the fatty degeneration of hepatic cells was relieved.Conclusion KDZOS can adjust the blood lipid metabolism in hyperlipoproteinemia rats and restrain fatty deposition in liver.

Abstract:Objective To investigate the anti-mutation of Chinese medicinal herb Herba Leonuri and its effect on T lymphocyte proliferation in spleen.Methods The micronucleus test of mouse bone marrow cell (MNT) :thirty mice were divided into six groups (n= 5)，negative control ( NS)，cyclophosphamide group (CP 3.0 mg•kg^-1)，Herba Leonuri antimutagenesis groups(Herba Leonuri with dosages of 1.0,2.0,4.0,8.0 g•kg^-1+CP30 mg•kg^-1).The improved method was used to detect the micronuclei frequency.Lymphocyte transformation test: twenty-four mice were divided into four groups (n=6)，saline control，CP control (30 mg•kg^-1)，Herba Leonuri (2.0 g•kg^-1)，Herba Leonuri +CP (2.0 g•kg^-1 Herba Leonuri +CP 30 mg•kg^-1). MTT assay was used to calculate the stimulation index (SI).Results The micronuclei frequencies in Herba Leonur 2.0,4.0,8.0 g•kg^-1 groups were lower than that in CP group (P<0.05);the SI in saline control, Herba Leonuri，Herba Leonuri +CP,CP groups were 1.89±0.19, 2.17±0.14, 1.73±0.13 and 1.45±0.09,respectively, the difference of SI was significant between Herba Leonuri and saline control groups (P<0.05); the difference of SI was significant between Herba Leonuri +CP and CP groups(P<0.05).Conclusion Herba Leonuri may protect genetic material from injury caused by CP, namely,showing effective antimutagenesis and yet Herba Leonuri could enhance lymphocyte function.

Abstract:Objective To study the effect of bone marrow mesenchymal stem cells (BMSCs) promoting the recovery of injured hepatic tissue and provide a new method for the treatment of liver diseases. Methods ①BMSCs were cultivated and amplified in vitro from bone marrow cell suspension of rats.Then the potential multi-differentiation of BMSCs was detected.②Healthy Wistar rats were randomly divided into experimental group (n=30) and control group (n=10).The hepati ischemia-reperfusion injury model was established.BMSCs were transplanted into ischemia-reperfusion livers of rats in experimental group via local transplantation and saline was transplanted into the rats in control group.The survival rate of rats was observed, as well as the recovery of liver.The mechanism of the reparation of hepatic injury was explored through observation of the trace of DAPI-labeling donator’[KG-*3]s BMSCs in acceptor’[KG-*3]s body. Results ①BMSCs not only had a high purification, homogeneity and stability, but also had unlimited proliferation.BMSCs could differentiate into osteoblast and adipocyte by being cultivated in induced media of osteoblast differentiation and adipocyte differentiation.②The survival rate of experimental group was 80%. The injured hepatic tissues were repaired.The blue fluorescent cells were found in the recovery of the host liver tissue.The survival rate of control group (non BMSCs implantation) was up to 30%.The survival rates of the two groups had a significant difference (P<0.05). Conclusion BMSCs are easily isolated and cultivated in vitro.BMSCs can improve hepatic ischemia-reperfusion injury via local transplantation.

differences in RBC,HB,HCT,MCV,MCH,MCHC,PLT,RDW,WBC,ALT,AST,ALP,T-BIL,GLU,ALB,TP,Cr,BUN betwen control group and pcDNA3-gB groups were not significant as well as the pathological results of liver,heart,kidney,brain,spleen,cornea,retina and trigeminal ganglion(P>0.05).Conclusion pcDNA3-gB with different doses have not significant effect on the indexes of hematology,hematological biochemistry and pathology in immunized mice.It is initially proved that pcDNA3-gB is safe.

Abstract:Objective To explore the damage effect of high level of glucose on ECV304 cells and the approaches in which high level of glucose plays its part to provide theoretical bases for the therapy of diabetes.Methods ECV304 cells were divided into four groups: normal control, high glucose group in which glucose was added to the cells with final concentration of 35 mmol•L^-1, Radix Astragali（RA） group in which glucose was added to the cells with final concentration of 35 mmol•L^-1, as well as RA with final concentration of 500 mg•L^-1, mannitol group in which mannitol was added to the cells with final concentration of 25 mmol•L^-1.The cells were cultivated for 24 h after the glucose, RA and mannitol were added to the cells and collected for the determination of intracellular Ca²⁺ concentration, mitochondrial membrane potential and the morphological changes of cells and mitochondria were observed under electronic microscope.Results The intracellular Ca²⁺ concentration in the cells of high glucose group was significantly higher than those of RA group, mannitol group and normal control group (P<0.05), the intracellular Ca²⁺ concentrations in the cells of RA group and mannitol group were significantly higher and the mitochondrial membrane potential was significantly lower than those of normal control group (P<0.05); the mitochondrial membrane potential in the cells of high glucose group was significantly lower than those of RA group, normal control group and mannitol group (P<0.05).The mitochondrial membrane potential in the cells of RA group was significantly higher than that of mannitol group ( P<0.05).The morphology of cells in RA group was normal, the mitochondrial structure was normal, the cristae in mitochondria were clear. The morphology of cells in high glucose group was abnormal, the mitochondria were swollen and the cristae were gone.Conclusion RA has protective effects on injury of ECV304 cells,especially mitochondrial injury,induced with high level of glucose.

Abstract:Objective To reconstruct the fibrin-based tissue engineering corneal epithelium using limbal stem cells as seed cells and provide new grafting material for corneal epithelium transplantation.Methods The fibrin-based frame was reconstructed by mixing 20 m•L^-1 fibrinogen solution and 10 U•mL^-1 lthrombin and the normal struture and ultrastructure of frame was detected. The limbus corneal tissue (2 mm×2 mm)was obtained,the fibrin-based frame after trypsind egestion.The growth status of the cells was obsewed ,and the changes of morphology,ultrastructure and immunohistochemistry were observed after two weeks. Results Fibrin-based frame was soft and had extensibility.Its dimension was 70-108 μm,the average empty porosity was 70.4%.It was multilayer network observed under scanning electron microscope.The cells migrated from the edge of the tissue 36 h after inoculation,single layer cells were formed after 7 d, the cells were combined with fibrin to form gragting implant after 14 d.The cells remained the typical ultrastructure feature of the epithelium observed under transmission electron microscope.The immunohistochemical results of the anti- cytokeratin McAb AE5 and the anti-p63 McAb 4A4 were positive. Conclusion Fibrin is a suitable frame material for corneal epithelium transplantation,provided a new grafting material for tissue engineering corneal epithelium.

Abstract:Objective To establish an animal model of isolated lung perfusion(ILP) system to evaluate the effects of the ILP chemotherapy with adriamycin(ADM) and the safety to the lung and body.Methods Total 16 rabbits were divided into 2 groups.One group(experiment group) was established the ILP system and carried the perfusion chemotherapy with ADM.One group(control group) was carried the normal vein chemotherapy with ADM.Then the ADM levels in the tissues of two lungs,heart,liver and kidney were examined by fluorescence spectrophotometry.The ratio of wet to dry lung weight was detected,and the pathological changes were observed after HE staining.Results The serum ADM levels were(0.055±0.125) mg•L^-1 in experiment group and (1.075±0.931) mg•L^-1 in control group,the serum ADM level in experiment group was lower than that in control group(P＜0.001).Compared with control group,the ADM level in the left lung increased(P＜0.001),but the ADM levels in the heart,liver,kidney decreased.The ratio of wet to dry weight of the left lung in experiment group was 6.491±0.248,and 5.937±0.361 in control group,there was no significant difference（P＞0.05）.No alveolar wall thicken,alveolar and mesenchyma edema were found in two groups.Conclusion ILP chemotherapy can get high drug concentration in the local lung tissue.It can decrease the toxicity to the whole-body and guarantee the safe of the perfusioin lung.It provide a safe and effective therapy for the advanced lung cancer and lung metastasis cancer.

Abstract:Objective To investigate the effect of implanted radioactive ¹²⁵I seeds on normal tissue structures of bronchus，esophagus,pulmonary artery,pulmonary vein and alveolus in dogs. Methods Nine healthy male dogs weighing 17-21 kg were randomly divided into three groups: 30 d,60 d experimental groups and control group.Radioactive ¹²⁵I seeds (3.7×10⁷ Bg,1.0 mCi) were implanted into the sides of bronchus,esophagus,pulmonary artery,pulmonary vein respectively,the samples of bronchus,esophagus,pulmonary artery,pulmonary vein were taken 30 and 60 d after transplantation,HE staining was used to observe the pathologic changes of the tissues under light microscope.Results The damages of normal bronchus,esophagus,pulmonary artery,pulmonary vein and alveolus after radioactive ¹²⁵I seeds implantation in 30 d group were weaker than those in control group and 60 d　 group,there were no complications such as perforation,hemorrhage,necrosis,etc.Histopathological score indicated that the scores of bronchus,esophagus and alveolar in 30 d group and 60 d group were higher than those in control group(P<0.05),there was no significant difference between 30 d group and 60 d group(P>0.05);there was no significant difference in histopathological score of pulmonary vein among all groups(P>0.05). Conclusion The implanted radioactive ¹²⁵I seeds can damage all kinds of tissues at different degrees,but this kind of damage is reversible,the dog may repair the damage through its own repair ability,its clinical application is safe.

To observe the inhibitory effect of peptide compound GHGKHKNK on invasion and metastasis of mouse melanoma cell line B16-F10,in order to exploit a drug with higher pertinency and independent intellectual property rights of our country. Methods Cytotoxic effect of GHGKHKNK on mouse melanoma B16-F10 malignant melanoma cells was evaluated by MTT assay; C57BL/6J mice the were used to establish experimental lung metastasis models by injecting,B16-F10 malignant melanoma cells into the lateral tail vein the effect of GHGKHKNK on experimental lung metastasis in mice was detected with immunohisto chemical analysis. Results The inhibitory of cell 17.4% after treated with 10^-1mol•L^-1 GHGKHKNK for 48 h. High dose of GHGKHKNK (500μg•kg^-1•d^-1)clearly suppressed lung metastasis in mice. Conclusion GHGKHKNK has a stronger inhibitory effect on invasion and metastasis of B16-F10 malignant melanoma cells.

To investigate the effect of insulin-like factor-I(IGF-Ⅰ)on the proliferation activity of human periodontal ligamental fibroblasts(PDLF).Methods The PDLF transfected with IGF-Ⅰ and PDLF untransfected with IGF-Ⅰ prepared in our lab were digested by 0.25% trypsin and prepared into cell suspension.The two sorts of cells were counted after being vaccinated, then the growth curves of the two cells were mapped and the growth speeds were compared.The PDLF transfected with IGF-Ⅰ and PDLF untransfected with IGF-Ⅰ were digested by 0.25% trypsin and then cultivated.The proliferation activities of the PDLF transfected with IGF-Ⅰ and PDLF untransfected with IGF-Ⅰ were observed using MTT methods.The activity of alkaline phosphatase(ALP) was determined by using ALP kit.Results The growth curves of the two cells showed that the growth speed of the PDLF transfected with IGF-Ⅰ was significantly faster than that of the PDLF untransfected with IGF-Ⅰ (P<0.05).The proliferation ability and ALP activity of the PDLF transfected with IGF-Ⅰ were both significantly higher than those of the PDLF untransfected with IGF-Ⅰ (P<0.05 or P<0.01). Conclusion IGF-Ⅰ may participate in the reconstruction of peridontal tissue through the function of PDLF.

To investigate the effects of membrane type-1 matrix metalloproteinase (MT1-MMP) on the biological behavior of breast carcinoma MCF-7 cells.Methods A eukaryotic expression vector pcDNA3.1 containing MT1-MMP and MT1-MMP-E240A was stably transfected into MCF-7 cells liposome transfection method.The expression level of MT1-MMP was detected by RT-PCR and immunofluorescence cytochemistry.The test of the growth in vitro, proliferation ability and migration in the extracellular matrix (ECM), as well as electron microscope observation were used to detect the biological behavior of MCF-7 cells after transfection.Results The expression of MT1-MMP mRNA of MCF-7 cells after transfection was increased compared with control group proved by RT-PCR,the expression of MT1-MMP protein was positive detected by immunofluorescence cytochemistry, the positive staining localized in the cytoplasm and cell membrane.Compared with control group,a lot of cells at division phase and tumor giant cells were found in MT1-MMP group and MT1-MMP-E240A group, glucogen pools and marrow body like lysosomes appeared in part of the cytoplasms observed under transmission electron microscope, The percentage of cells at G₂M phase was increased in MT1-MMP group and MT1-MMP-E240A group compared with control group（P＜0.01）.The migration ability of transfected cells was also increased on the Matrigel and Fibronectin compared with control group（P＜0.05）. Conclusion MT1-MMP can promote the proliferation, migration of MCF-7 cells and increase their malignant phenotype.MT1-MMP catalytic domain does not play prominent role in the proliferation, migration and malignant phenotype of MCF-7 cells.

To investigate the genentic association between the peroxisome proliferators-activated receptor γ (PPARγ) gene polymorphism and schizophrenia in the Han descent population in the north of China.Methods A PCR-based RFLP procedure was employed to detect the alleles and genotypes of PPARγ gene in 111family trios including 333 subjects of Han descent population in the north of China. Results No significant difference was found in the frequencies of genotypes and alleles of the two SNPs (rs2972162 and rs2938395) of PPARγ gene between case group and control group.The TDT analysis result showed that the two SNPs were not associated with schizophrenia.The 2-SNP haplotype was not associated with schizophrenia.Conclusion There is no genentic association between the PPARγ gene polymorphism and schizophrenia in the Han descent population in the north of China.

To investigate the genetic association between the polymorphism of rs3814500 site on PTGDS gene and schizophrenia in Han population from the north of China.Methods PCR-RFLP analysis was employed to detect the genotype of PTGDS gene in 141 family trios consisting of fathers,mothers and affected offsprings with schizophrenia.Haplotype relative risk(HRR) analysis and transmission disequilibrium test(TDT) were used to analysis the genotype data.Results The genotypic frequency of the PTGDS gene did not deviate from Hardy-Weinberg equilibrium in both patient group and parent group(P>0.05); HRR analysis did not show allelic association with the PTGDS gene(χ²=0.095, P>0.05);TDT analysis did not show preferential transmission of the two alleles（χ²=0.103，P>0.05）. Conclusion There is no genetic association between the polymorphism of rs3814500 site on PTGDS gene and schizophrenia in Han population from the north of China.

To study the relationship between heat shock protein 70 (HSP70) and the pathogenesis of peptic ulcer disease, acute erosive hemorrhagic gastritis. Methods Three groups were selected by conducting endoscopy: 75 patients with peptic ulcer at active stage (29 cases of gastric ulcer, 46 cases of duodenal ulcer), 13 patients 　with acute erosive hemorrhagic gastritis, 26 cases of normal gastric mucosa normal, and the patients in the first two groups were given antiulcer therapy.The expressions of HSP70 and GAPDH mRNA before and after treatment were detected by reverse transcription polymerase chain reaction (RT-PCR). Results The expression level of HSP70 mRNA in ulcer group was significantly higher than that in normal group (P<0.01).The expression levels of HSP70 mRNA in ulcer group at three stages (active, healing, and scar stages)after treatment were significantly higher than those before treatment respectively(P< 0.01 or P<0.05).Nevertheless,after treatment, there were no significant differences between various stages(active, healing, and scarring stage) of ulcer group and also between various periods(one month, two months) at each stage(P>0.05).The expression level of HSP70 mRNA in gastritis group was higher than that in normal group(P<0.01).In gastritis group(improved group and healing group),the expression levels of HSP70 mRNA one month after treatment were higher than those before treatment(P<0.01 or P<0.05).Conclusion HSP70 as a stress protein,participates in repair of peptic ulcer and acute erosive hamorrhagic gastritis.

To detect the mutation of the acid-resistant gene dgk in the fluoride-resistant strain of Streptococcus mutans and to suppose the significance of the gene mutation. Methods Fluoride-resistant strain Ingbritt-FR was obtained by subculturing S treptococcus.mutans Ingbritt to TSA and TSB containing different concentrations of fluoride.Primers was designed according to dgk’s sequence of S treptococus. mutans from GenBank database and PCR was perform. The acquired sequence was connected with PGEM-T vector and sequenced. Results The fragment was acquired by PCR according with the expected results. The dgk’s sequence had 8 changed bases, a A→T transition in codon 904,a G→C transition in codon 940,a T→C transition in codon 946,a C→T transition in codon 952,a C→T transition in codon 958,a T→C transition in codon 964,a G→A transition in codon 976 and a G→A transition in codon 991 were observed.The result was submitted to American GenBank,and the accession number is DQ272517.Conclusion The mutation in theacid-resistant gene dgk of the fluoride-resistant strain of Streptococcus mutans is point mutation and samesense mutation.

To detect the cell cycle change and the apoptosis at each phase of cell cycle in laryngeal carcinoma cells,and discuss the role of the cell cycle change and the apoptosis at each phase of cell cycle in the occurrence,development and prognosis of laryngeal carcinoma.Methods The TUNEL technique and flow cytometry(FCM) parameter analysis were used to detect the cell cycle change and the apoptosis at each phase of cell cycle in 15 cases of polyp of vocal cord and 387 patients with laryngeal carcinoma.Results Compared with the group of polyp of vocal cord,although the proportion of the cells at G₀G₁ phase increased in the group of laryngeal carcinoma,there was no significant difference(P>0.05).The apoptotic index (AI) in laryngeal carcinoma was obviously lower than that in polyp of vocal cord,and this phenomenon was mainly caused by the decrease of the apoptosis of the cells at G₀G₁ phase.The proportion of the cells at G₂M phase,the total AI,the AI at S phase and G₂M phase were obviously enhanced in the poor-differentiated cancer,and this result was also observed both in the group of recurrence and in the patients who died in 5 years after operation,there was no significant difference between the well-differentiated and the moderate-differentiated cancer.The proportion of the cells at G₂M phase,the total AI and the AI at G₀G₁ phase in the group of no-relapse and the patients who lived more than 5 years after operation were strongly lower than that in polyp of vocal cord(P>0.05).Conclusion The significant increasing of the proportion of the cells at G₂M phase,the total AI,the AI at S phase and G₂M phase could be regard as S indicators to judge prognosis and assistant therapy for laryngeal carcinoma.

To study the levels of matrix metalloproteinase-3(MMP-3),matrix metalloproteinase-9(MMP-9) and inhibitor of metalloproteinases-1(TIMP-1) in patients with acute myocardial infarction(AMI) and mechanism of plaque rupture of coronary atherosclerosis.Methods Sixty-seven patients with AMI were selected,average invasion time was 3 h～5 d.40 cases of non-coronary heart disease were included in control group.The expressions of serum MMP-3,MMP-9,TIMP-1 and the levels of serum cTnI,CK-MB were analyzed by enzyme-linked immunosorbent assay. Results The serum MMP-3,MMP-9 and TIMP-1 levels in AMI group were higher than those in control group,there was significant difference between two groups（P<0.05 or P<0.01 ）.MMP-3/TIMP-1 and MMP-9/TIMP-1 in AMI group were higher than those in control group （P<0.05）.There was no correlation of serum MMP-3,MMP-9 and TIMP-1 with the levels of cTnI and CK-MB.Conclusion The overexpressions of MMP-3,MMP-9 and MMP-3/TIMP-1,MMP-9/TIMP-1 disorder may be involved in the procession of plaque rupture of coronary atherosclerosis and AMI.

To investigate the effect of cyclooxygenase-2(COX-2) on angiogenesis through detecting the expressions of COX-2 and vascular endothelial growth factor (VEGF) in hepatic cell carcinoma (HCC) tissues.Methods The expressions of COX-2 and VEGF in 30 cases of HCC and 10 normal liver tissues were detected by immunohistochemistry (SP).At the same time,the new vessels were marked with CD34, the tumor microvessel density(MVD) was determined by counting the number of CD34 positive cells of blood vessel endothelium.Results The　 expressions of COX-2 and VEGF in HCC tissues were significant higher than those in normal liver tissues(P<0.001).There was significant positive correlation between the expressions of COX-2 and VEGF (r=0.632,P<0.01).MVD in the positive expression tissues of COX-2 and VEGF was significant higher than that in both negative tissues(P<0.01). Conclusion The high expression of VEGF is significantly correlated with the expression of COX-2,it indicates that COX-2 can induce VEGF expression and promote angiogenesis in HCC.

To research the relationship between hypoxia-inducible factor-1 alpha(HIF-lα) expression and the recurrence of differentiated thyroid carcinoma(DTC) after complete thyroidectomy combined with ¹³¹I treatment，and explore its possible mechanism of recurrence. Methods The recurrence rate of 173 cases of DTC after complete thyroidectomy within 3 years was analyzed,according to the follow-up detecting data of Tg,TgAb,B-mode ultrasonography,X-ray scanning and global ¹³¹I imaging,the cases of recurrence were used as recurrence group,the same number cases without recurrence as control group which were chosen randomly.The HIF-lα expressions in recurrence cancer tissues and un-recurrence cancer tissues were detected with immunohistochemical staining and RT-PCR technique. Results The 3-year recurrence rate after thyroidectomy was 5.20%(9/173),among them, the recurrence rate of papillary carcinoma was 4.90%(5/102),while the recurrence rate of follicle carcinoma was 5.63%(4/71),there was no significant difference between two groups (P>0.05).The HIF-lα expression levels in recurrence tissues of papillary carcinoma and follicle carcinoma were significantly higher than those in unrecurrence tissues (P<0.05).Conclusion Several recurrence cases can be still found in DTC cases after complete thyroidectomy combined with ¹³¹I treatment,one of its mechanisms may be related to significant increase of the HIF-lα expression level in cancer tissue.

To investigate the role of the mass screening by comparing the pathological features of prostate cancer between mass screening patients and clinical patients. Methods 107 cases of prostate cancer(including 51 patients from clinical diagnosis and 56 patients from mass screening) and 7 cases of prostate intraepithelial neoplasia(PIN, from mass screening) were analyzed using the Gleason’s grade system. Results ① Gleason’s grade of prostate cancer in mass screening group was lower than that in clinical diagnosis group(χ² =48.22, P<0.001); ② Gleason’s scores of prostate cancer in mass screening group were lower than those in clinical diagnosis group(χ² =24.55, P<0.001); ③ Differentiated degrees of prostate cancer in mass screening group were higher than those in clinical diagnosis group(χ² =22.46, P<0.001); ④ The typical PIN was found only in mass screening group. Conclusion In mass screening group the moderately differentiated carcinoma is the most common type of prostate cancer, but in clinical diagnosis group the poorly differentiated carcinoma is most. General investigation for prostate cancer is benefit to find caner of early stage.

To evaluate the effects of patient controlled analgesia（PCEA）on the perioperative changes of circulatory and pulmonary function of elderly with hypertensions after abdominal surgery. Methods Twenty-eight patients of ASAⅡ-Ⅲ aged more than 60 years undergoing uratomy were randomly divided into two groups: control group and PCEA group.Preoperative and postoperative circulatory and pulmonary functions were measured with noninvasion circulatory monitor and pocket lung function meter respectively.Results In control group，the systolic pressure，diastolic pressure，and heart rate increased by 19%,17% and 19%,respectively, as compared with preoperation.The percentage of forced vital capacity (FVC%),percentage of forced expiratory volume in first second to forced vital capacity(FEV1%) and percentage of maximal ventilatory volume (MVV%) of postoperation in control group were significantly decreased compared with preoperation （P<0.05）.Whereas,in PCEA group，the blood pressure，heart rate，and pulmonary parameter had no significant change between preoperation and postoperation,but the blood pressure and heart rate were significantly lower than those in control group(P<0.05).In PCEA group,except MVV%,the other pulmonary parameters had no obviously change as compared with preoperation,but they were all better than those in control group(P<0.05). Conclusion Postoperation PCEA can provide a better analgesic effect and improve the function of circulatory and respiratory system,and also reduce the risk of the operation.

To reveal the geographical distribution regularity of the normal reference value of old female’s　 hemoglobin in China,and provide scientific basis for making its unified standards.Methods The normal reference values of 15 690 examples of old female’s hematocrit by the method of hemoglobin cyanide(HiCN)and five geographical factors in 157 areas in China,its distribution regularity and the complex relationship characteristic between geography environment elements were analyzed with the methods of factor analysis,spatial analysis of GIS and correlation analysis.Results By the method of factor analysis,the five factors of geography environment were combined into two common factors F₁and F₂;the original data was replaced with its scores to infer the regression equation :y^＝127.9－0.01009F₁－0.01543F₂±8.8.And this equation was used to figure out the reference value of 1 288 observation points in China . With support of the model of spatial analysis of GIS,the distribution regulation of old female’s hemoglobin reference value in China and its distribution regularity was west-higher,east-lower,north-higher and south-lower. Conclusion If some area’[KG-*3]s altitude (X₁),sunshine hours yearly(X₂),mean relative humidity yearly (X₃),mean temperature yearly(X₄),precipitation yearly (X₅) are known,this place’[KG-*3]s reference value of hemoglobin can be calculated out by using this equation.The reference value of hemoglobin in any areas in China can be gotten from the trend distribution regularity map.

To evaluate the water quality of Chagan Lake by analyzing 15 metal elements and 15 rare earth elements (REE) contents in rainfall season and rainless season of Chagan Lake of Jilin province.Methods In　 accordance with the relevant national standards,ICP-MS,ICP-AES,AAS and AFS were performed to determine the water guality of Chagan Lake in rainfall season and rainless season,the data of rainfall season and rainless was compared.Results The metal element contents in rainfall season and rainless season were lower than the water quality standard of class Ⅲ　Surface Water Quality Standards in China.The variation of the con tents of Hg,Ba,U in rainfull season compared with rainless season was not significant（P>0.05）,the contents of the remaining elements in the rainfall season were slightly higher than those in rainless season（P>0.05）;the contents o f Be,Co,Ni,Cu,Zn,Se,Pb in rainfall season were higher than those in rainless season and had statistically significant difference (P<0.05). Conclusion The water quality state of Chagan Lake is good,and it is unease to cause the poison of As,Hg,Pb and other heavy metals induced by of bioaccumulation role of the food chain ,the result shows that Chagan Lake wetland areas have not been polluted with heavy industrial pollution sources.