Objective To study the effects of IL-24 gene combined with ionizing radiation on apoptosis in PC-3 cell line in order to prepare the ground for combined therapy of IL-24 gene and ionzing raditaion for tumor.Methods The 　experiment was divided into sham irradiation group and irradiation groups with different irradiation doses,which were 2,4,6,8,12 and 18 Gy, respectively.To detect the expression of IL-24 gene,three groups were included,which were control group (1×PBS),vector group and IL-24 gene group.To detect the apoptotic effect of IL-24 gene combined with ionization radiation on PC-3 cell line,the experiment was divided into control group,vector group,IL-24 gene group,irradiation group (6 Gy),vector combined with irradiation group and IL-24 gene combined with irradiation group.Alkaline lysis assay was used to extract and purify the plasmid.Plasmids were transfected into PC-3 cell line by polyethyleneimine（PEI）in vitro.The expression of the interest gene was detected by RT-PCR.The changes of apoptosis in PC-3 cell line were detected by flow cytometry（FCM）using the staining of Annexin-V and PI.Results Compared with sham-irradiation group,the apoptotic percentage of PC-3 cell line did not show marked change after 2 and 4 Gy X-rays irradiation for 48 h（P>0.05）.The apoptotic percentage was increased significantly after X-rays irradiation with the dose of 6 Gy（P<0.01），and the mean apoptotic percentage of PC-3 cell line was increased by a factor of 1.6 to 3.0 compared with sham-irradiation group.The expression of IL-24 gene could be observed in PC-3 cell line transfected by IL-24 gene except in control group and vector group，and all of them showed the expression of GAPDH gene.As compared with the other groups,the number of early apoptotic cells of PC-3 cell line in the IL-24 gene combined with irradiation group was increased significantly （P<0.05） except in irradiation roup.The number of late apoptotic and necrotic cells of PC-3 cell line in the IL-24 gene combined with irradiation group was increased significantly compared with control group,irradiation group and vector combined with irradiation group（P<0.05,P<0.01）.As compared with the other groups,the total number of apoptotic and necrotic cells in the IL-24 gene combined with irradiation group was increased significantly （P<0.05，P<0.01） except in IL-24 gene group.Conclusion IL-24 gene combined with X-rays irradiation could induce effective apoptosis in PC-3 cell line.

Objective To explore the changes of spleen lymphocyte subgroups in diabetic rats after multiple low dose radiation(LDR).Methods The experiment was divided into normal control group,pure diabetes mellitus(DM) group,and DM plus different doses of irradiation groups ( the irradiation doses were 0.025,0.050 and 0.075 Gy,respectively).The diabetic rat model was induced by intraperitoneal injection of streptozotocin.After the diabetic rats were irradiated 15 times,the percentages of spleen CD4⁺ and CD8⁺ T cells and ratio of CD4⁺/CD8⁺ T cells were detected with flow cytometry on the fourth weekend.Results The diabetic rats manifested obvious polydipsia,polyphagia,polyuria and weight loss.On the fourth weekend after irradiation,as compared with normal control group,the percentage of spleen CD4⁺ T cells increased significantly (P<0.01),and that of CD8⁺ T cells decreased significantly (P<0.01),and the ratio of CD4⁺/CD8⁺ T cells was increased significantly (P<0.01) in diabetic rats; but the changes of spleen lymphocyte subgroups were not significant in LDR plus DM groups.As compared with DM group,the percentages of spleen CD4⁺ T cells were declined markedly in both 0.050 and 0.075 Gy plus DM groups (P<0.05,P<0.01 ),and those of CD8⁺ T cells increased significantly in LDR plus DM groups(P<0.01),and the ratio of CD4⁺/CD8⁺ T cells was declined obviously (P<0.01).Conclusion The　 multiple LDR could regulate the immune function in diabetic rats,and rectificate the immunological imbalance in order to protect body.

Objective To evaluate the antitumor efficiency of IL-12 gene mediated by hydrodynamic injection in mouse models of malignant ascites.Methods To prepare the malignant ascites model,H₂₂ hepatoma cells were injected into peritoneal cavity of mice.The mice were divided into 3 groups.Plasmid pCMV-mIL-12,pCMVβ and saline were respectively administered into mice by hydrodynamic injection 3 d after H₂₂ cells injection.Thereafter,the blood was collected at indicated time points.The expressions of IL-12 and IFN-γ and NO in serum were determined.Three mice were sacrificed 14 d after treatment.The volume of ascites was measured.The cells in ascites were stained with hematoxylin and eosin or AO/EB.The other mice were kept to monitor the survival.Results Compared with mice treated with pCMVβ or saline,the survival of mice treated with pCMV-mIL-12 was obviously prolonged (P<0.01).The significant elevation of IL-12 and IFN-γ and NO levels in serum were detected(P<0.01,P<0.05) and less alive tumor cells(P<0.01),more apoptotic cells (P<0.01) were observed in mice treated with pCMV-mIL-12. Conclusion Hydrodynamic injection-mediated IL-12 gene transfer can prolong the survival of mice bearing malignant ascites by decreasing the production of hemorrhagic ascites and inducing apoptosis of tumor cells.

Objective To investigate the protective effect of gross saponin tribulus terrestris (GSTT) on the apoptosis of pheochromocytoma cells (PC12 cells )induced by H₂O₂ and its mechanisms.Methods PC12 cells were divided into control,model,high dose GSTT(GSTT₁) and low dose GSTT(GSTT₂) groups.Apoptosis of PC12 cells was induced by H₂O₂ at the concentration of 300μmmol•L^-1.The cell activity was determined by MTT.The subdiploid peaks showing cell apoptosis rate and △Ψm were detected by flow cytometry.Proteins of Bcl-2 and Bax were detected by immunohistochemistry.Results Compared with model group,the survival rate of PC12 increased （P<0.001),the apoptotic rate and protein expression of Bax decreased（P<0.01),and the △Ψm and protein expression of Bcl-2 increased with the raise of GSTT dose(P<0.05).Conclusion GSTT can inhibit the apoptosis of PC12 induced by H₂O₂ which might be correlated with the inhibition of apoptosis in the path of mitochondrion.

Objective To study the condition of pheochromocytoma cells(PC12 cells) differentiated into neuron-like cells induced by retinoic acid (RA) and to observe the lengh of neurite and max diameter and the expression of MAP2 in PC12 cells during their differentiation into neuron-like cells. Methods There were seven groups : 0.1,0.3,0.5,1.0,2.0 and 5.0 mg•L^-1RA groups and control group which contained 10% fetal calf serum.PC12 cells were exposed to RA with different concentrations for 72 h and the lengh of neurite and max diameter of PC12 cells were observed at 24,48,and 72 h respectively. The expression of MAP2 was detected by immunocytochemistry after PC12 cells were exposed to RA for 72 h.Results The number of MAP2 positive cells was significantly increased in 0.3,0.5,1.0,2.0 mg•L^-1RA groups compared with control group and the most significant concentration was 1.0 mg•L^-1（P<0.05）.The length of total primary neurite and max diameter of PC12 cells in 0.3,0.5,1.0,2.0 mg•L^-1RA groups were obviously longer and bigger than those in control group and the most significant concentration also was 1.0 mg•L^-1（P<0.01）.But the number and the lengh of neurite were fewer and shorter when PC12 cells were exposed to 2.0 mg•L^-1RA　 than that of 1.0 mg•L^-1 RA.There were a lot of black particles in PC12 cells and cells became smaller and aging when they were exposed to 2.0 mg•L^-1RA　 for 7 d. Conclusion PC12 cells could differentiate into neuron-like in vitro induced by RA,and RA could enhance the outgrowth of neurite and multiplication of max diameter of the differentiated PC12 cells.1.0 mg•L^-1 is 　the optimal concentration in which RA could induce PC12 cells to differentiate into neuron-like cells.

Objective To study the effect of dopamine on the epithelial sodium channel (ENaC) in conscious euvolumic rats.Methods Animals were divided into two groups: experimental group (n=29) and control group (n=16).In conscious rats,the body sodium and fluid balance were constantly maintained by servo-controlled replacements,and mean arterial blood pressure (MAP),urine volume,sodium excretion and glomerular filtration rate (GFR) were determined in response to continuous dopamine infusion (5 μg^-1•kg^-1•min^-1),and the expressions of ENaC subunits (αENaC,βENaC,and γENaC)in kidney tissues were analyzed by immunoblotting method.Results Compared with control group,the MAP and GFR in experimental group did not change(P>0.05),but urine volume and urine sodium concentration increased significantly(P<0.05);the expressions of αENaC and βENaC did not change,but the expression of γENaC was decreased(P<0.05).Conclusion Dopamine　 infusion induces a sustained natriuresis and diuresis without influence on MAP and GFR,and its mechanism may be related to down-regulation of the expression of γENaC.

Objective Abstract：Objective To explore the effects of arsernic trioxide (As₂O₃) on telomerase activity and cellular apoptosis of human colon cancer cell line CCL-187.Methods CCL-187 cells were divided into control group，1.0 μmol•L^-1As₂O₃ group and 2.5 μmol•L^-1As₂O₃ group，all groups were cultivated for 1 to 6 d.The inhitory rate of cell growth was detected by MTT assay.The morphologic changes of CCL-187 cells were observed under electron microscope.The expression of hTERT at mRNA level was analyzed by RT-PCR.Telomerase activity was determined by TRAP-PCR-ELISA.Results The differentiation of cells was found in 1.0　μmol•L^-1 As₂O₃ group，but apoptosis in 2.5　μmol•L^-1 As₂O₃ group，and at the same time ，the telomerase activity was reduced and the hTERT-mRNA expression was downregulate in 1.0 μmol•L^-1 As₂O₃ group and 2.5 μmol•L^-1 As₂O₃ group in a time-dependent manner.There were significant differences compated with control group（P<0.01 or P<0.01）;there were also significant differences between 2.5 μmol•L^-1 As₂O₃ group and 1.0 μmol•L^-1 As₂O₃ group（P<0.01）.The inhibitory rate of cell growth was closely negatively correlated with the downregulation of hTERT-mRNA（1.0 μmol•L^-1 As₂O₃ group,r=－0.803，P<0.01；2.5 μmol•L^-1 As₂O₃ group,r=－0.697，P<0.01） and the reduction of telomerase activity（1.0 μmol•L^-1 As₂O₃ group,r=－0.836， P<0.01；2.5 μmol•L^-1As₂O₃ group,r=－0.792，P<0.01）.The reduction of telomerase activity was closely correlated with the downregulation of the hTERT-mRNA（1.0 μmol•L^-1 As₂O₃ group,r=0.956，P<0.01； 2.5 μmol•L^-1 As₂O₃ group,r=0.988，P<0.01）.Conclusion As₂O₃ can induce differentiation and apoptosis of CCL-187 cells.Its mechnism may be ralated to reducting the telomerase activity through downregulation of the hTERT-mRNA expression.

Objective To investigate the effects of geldanamycin (GDM) on the proliferation and relative gene expression in glioma cells induced by hepatocyte growth factor (HGF).Methods U251 MG and U87 MG of human malignant glioma cells were cultivated by cell culture technique.After cultivated in the presence of HGF for 24 h,U251 MG and U87 MG cells were cultivated for another 48 h in the presence of GDM at different concentrations (50,250,500, and 1 000 nmol•L^-1).The inhibitory rates of growth were examined by MTT essays.The experiment was divided into normal cells,cells+HGF (30 μg•L^-1),cells+GDM (50,250,500 and 1 000 nmol•L^-1　 and cells+HGF+GDM and paclitaxel groups.The relative gene mRNA expression level was detected with RT-PCR methods.Results After U251 MG and U87 MG cells were treated with HGF for 24 h,the proliferative rates were 0.139±0.07 and 0.242±0.167, respectively;and 50,250,500 and 1 000 nmol•L^-1 GDM had inhibitory effects on U251 and U87 cells,the inhibitory rates were 0.029±0.028,0.027±0.017,0.312 ±0.084,0.339±0.047 and 0.116±0.069,0.222±0.191,0.269±0.056,0.276±0.031; the inhibitory rates of paclitaxel on U251 MG and U87 MG cells were 0.075±0.062 and 0.071±0.044.The results of RT-PCR appered that the HGF and c-Met mRNA in U251 MG and U87 MG cells in HGF group were lower than those in normal group(（P<0.05）,but the HGF and c-met mRNA in U251 MG and U87 MG cells in HGF+GDM group were lower than those in HGF group (P<0.05).Conclusion GDM could inhibit the proliferation of glioma cells induced by HGF and regulate the relative gene expression.

Objective To study the effects of ginseng fruit saponins(GFS) on hemodynamics in acute myocardial infarction dogs.Methods The acute myocardial infarction models were induced by ligation left anterior descending coronary for 6 h in 30 dogs.GFS was divided into three doses groups(2.5,5.0 and 10.0 mg•kg^-1) and administered with intravenous drip.Pulse-activating injection was taken as positive control medicine and administered with intravenous drip.Infarction model group, normal saline was administered with intravenous drip.After treated with GFS (in dosages of 2.5-10 mg•kg^-1 iv drip )for 90 min,the changes of parameters of heart function and hemodynamics were recorded.Results Compared with model control group,the heart rate (HR) was slowed (P<0.05 or P<0.01); systolic blood pressure (SBP),diastolic blood pressure (DBP),left ventricular systolic pressure (LVSP),maximum left ventricular rise rate (+dp/dt_max) and maximum left ventricular fall rate (－dp/dt_max) and cardiac output (CO) were increased (P<0.05 or P<0.01) ; and left ventricular end-diastolic pressure (LVEDP) was decreased after treated with GFS (in dosages of 5.0 and 10.0 mg•kg^-1 iv drip) for 90 min. Its effect was the same with pulse-activating injection.Conclusion GFS can improve myocardial systolic and diastolic function,and relieve pump failure after myocardial infarction.GFS can also decrease the myocardial volume of oxygen consumption to profit increasing myocardial blood-supply.

Objective To discuss the effect of total arasolides of aralia elata（Miq） seem (TASAES) on cardiac function and protective effect on mgocardial structure in rats with early stage diabetes． Methods The models of diabetic rats were made by an intra-abdominal injection of streptozotocin(STZ)．Rats were divided into control group（C group），model group（M group） and TASAES 4.9,9.8,19.6 mg•kg^-1 groups（T₁，T₂，T₃ groups）．At the time of 8 weeks, the hemodynamic parameters were detected in various groups; ultrastructure of cardiomyocytes was observed under electron microscope；the expression of connective tissue growth factor(CTGF) was detected by RT-PCR technique at the molecular level．Results Compared with model group,TASAES increased the absolute value of left ventricular systolic pressure（LVSP） and ±dp/dt_max in diabetic rats，especially in high and middle dosage groups (P<0.05，P<0.01),it improved the ultrastructure of cardiomyocytes and reduced the expression of CTGF in high and middle dosage groups (P<0.01).Conclusion TASAES can improve the cardiac function　of diabetic rats in a dose dependent manner， and its protective effect may be related to the reduction of CTGF and enhancement of cardiac contractility.

Objective To explore the effects and central mechanisms of oxymatrine(OMT) on injury induced by focal cerebral ischemia reperfusion in rats.Methods The focal cerebral ischemia reperfusion was induced by middle cerebral artery occlusion in rats.The rats injected by intraperitoneal were randomly divided into sham group,focal cerebral ischemia reperfusion model group,LLT 31.25 mg•kg^-1 group,and OMT 35,70 and 105 mg•kg^-1 groups.The rats injected by cerebral ventricle were randomly divided into sham group,focal cerebral ischemia reperfusion model group,LLT 0.15 mg•kg^-1 group,and OMT 0.35 mg•kg^-1 group.The neurological score and infarct areas were used to evaluate the protective effects of OMT in focal cerebral ischemia reperfusion rats.The NO content in serum was determined in focal cerebral ischemia reperfusion rats injected by cerebral ventricle.Results OMT 70 and 105 mg•kg^-1 by intraperitoneal injection decreased neurological score（P<0.05）,deflated infarct areas(P<0.05) compared with focal cerebral ischemia reperfusion model group.Compared with focal cerebral ischemia reperfusion model group,the infarct area and the NO content in serum were decreased in rats treated with OMT 0.35 mg•kg^-1 by cerebral ventricle injection.Conclusion OMT injected by cerebral ventricle has protection against injury induced by cerebral ischemia reperfusion in rats.The central mechanism may be one of mechanisms of OMT.

Objective To express the several linked antigen epitopes of consence genes of HIV-1gp41/gp120 and HIV-2gp125/gp36 in the system of prokaryotic expression vector;to purify and identify the expression products.Methods In order to construct the recombinant expression plasmid pRSETB-env,the linked genes contained three antigen epitopes of gp41,two antigen epitopes of gp120,one antigen epitope of gp36 and three antigen epitopes of gp125 were inserted into pRSETB vector.After transformed into E.coli BL21(DE3) cells,the recombinant protein was expressed with induction of isopropy1β-D-thiogalactopyranoside(IPTG),and was purified by immobilized metalion affinity chromatograph and was recoveried by decreasing the concentration of urea.The immunoactivity was analyzed by Western blotting and enzyme-linked immunosorbent assay (ELISA).Results An expected expressing protein band (about 44 000) was seen with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The aim genes revealed higher expressive rate in E.coli BL21(DE3) cells.The results of Western blotting and ELISA showed specific reactions with HIV patients’sera,and no cross-reaction with other patients’sera using the expression protein.Conclusion The recombinant expression plasmid pRSETB-env which contained the several linked antigen epitopes of envelop genes of HIV is successfully constructed.After purification,the expression protein possesses higher pureness,good specificity and activity.

Objective To obtain ScFv gene from ovarian cancer patients with gene recombinant technique and express it into pichia pastoris in order to provide experimental basis of application of ScFv in diagnosis and treatment of ovarian cancer.Methods The total RNA of lymphocytes cell was extracted from ovarian cancer patients by TRIZOL.V_H and V_L gene were obtained by SOEingPCR.pPICZa/ScFv was contructed and transformed into X-33 cells.The expression was induced by pichia pastoris.The engineer bacteria with high effective expression of ScFv was screened. Results ScFv gene was constructed successfully.ScFv gene was about 700 bp and protein was induced by pichia pastoris and it was identified that its molecular weight was about 26 000.Conclusion ScFv gene, pPICZα/ScFv and pichia pastoris engineer bacteria with high effective expression of ScFv are obtained.

Objective To observe the protective effect of progesterone(PROG) on rat retinal ganglion cells(RGCs) in chronic ocular hypertention models.Methods The chronic ocular hypertention rat model was made by cauterizating three episcleral veins.Rats were divided into control group,high level PROG group,middle level PROG group,low level PROG group according to different concentrations of PROG injected intraperitoneally.The left eye was model eye and the right eye was control eye.Three months later,the animals were executed and the eyeballs were enucleated.The RGCs were detected by HE staining and Thy-1.1 immunohistological staining.The apoptosis of RGCs was detected by TdT-dUDP terminal nick end-labeling (TUNEL) technique.Results The number of RGCs of model eyes in high level PROG injection group was more than those in control group,low level and middle level groups(P<0.05).And the number of TUNEL-positive cells in high level PROG group was less than those in control group,low level and middle level groups(P<0.05).Conclusion PROG has neuroprotection on RGCs in rat chronic glaucoma model and the neuroprotection may be connected with concentrations of PROG.

Objective To study the relationship between hyperhomocysteic acid(HHcy) and artherosclerosis.Methods 20 male rabbits were randomly divided into control and experiment groups(n=10).HHcy models were made by hypodermical injection of DL-methionine for 7 weeks.After 7 weeks homocysteic acid(Hcy),IL-1β,IL-6,and IL-8 in serum of rabbits in two groups were measured by enzyme linked immunosorbent assay(ELISA).And the pathomorphological changes of aorta were observed with HE staining.The number of NF-κB positive cells in the aorta were counted by immunohistochemical method.Results The levels of Hcy,IL-1β,IL-6 and IL-8 in experiment group were higher than those in control group (P<0.05).Artherosclerosis changes were found in the aorta in experiment group and the number of NF-κB positive cells in the aorta in experiment group were more than those in control group（all P<0.05）.Conclusion The excessive secretion of inflammative factors is one of the mechanisms that HHcy causes artherosclerosis and NF-κB plays an importment mediation role.

Objective To investigate the expression of cycooxygenase 2(COX2) in myocardial fibrosis and significant.Methods Rat myocardium necrosis model was copyed by isoproterenol(Iso)injection (15 mg•kg^-1).The serum aspartate transaminase (AST),lactate dehydrogenase (LDH),creatine kinase (CK) and creatine kinase isoenzymes (CK-MB) in rats were detected by MD-100 automatic biochemical analyzer. The expression of COX2 protein was analyzed by immunohistochemistry. The expression of COX2 mRNA was analyzed by RT-PCR.Results Compared　 with control group,the levels of serum LDH,CK and CK-MB reached peak at 4 h (P<0.05),AST achieved peak at 6 h (P<0.05). The protein expression of COX2 was increased according with the fibrosis severity.COX2 mRNA arrived peak at 24 h (P<0.05) and began decreased at 48 h,but still higher than normal level (P<0.05).Conclusion COX2 mRNA and protein expression levels are increased significantly in myocardial ischemia and the increasing of COX2 expression has a closed relation with fibrosis,which indicates COX2 may be involved in the process of myocardial fibrosis.

Objective To investigate the anti-tumor effects and mode action of Apoptin on human uterine cervix cancer cells. Methods Recombinant plasmid pVAX1-Apoptin and pVAX1 were transfected into Hela cells by application of liposome in vitro and used as pVAX1-Apoptin group and pVAX1 group, meanwhile control group(without cells) was set up. The expression of Apoptin in Hela cells was detected by Western blotting. Anti-tumor effect on Hela cells was measured through methyl thiazolyl tetrazolium (MTT) assay. The alteration of mitochondrial transmembrane potential and ROS level of the cells were detected by flow cytometry ( FCM) with rhodamine 123 and DCFA staining. The activation of caspase-3 was assayed by its substrate color reaction. Results The　 inhibitory rate in pVAX1-Apoptin group 48 h after transfection (69.28%) was higher than those in control group (0.74%) and pVAX1 group(10.11%) .Compared with control group ,the mitochondrial transmembrane potential was decreased (P<0.01), ROS level of the cells was increased (P<0.05), and caspase-3 activity was increased(P<0.01) in pVAX1-Apoptin group.Conclusion Anti-tumor effects of Apoptin on Hela cells may be resulted from the apoptosis-inducing function of Apoptin.

Objective To observe the immune responses stimulated by inoculation of DNA vaccine encoding a fusion protein of CEA tandem repeat epitopes and heterogenous HSP70 fragment as a basis for pioneering a novol DNA vaccine specific to carcinomas.Methods The recombinant sequence for two CEA epitopes was tandemly engaged and inserted into-upstream of in-frame-varied HSP70 of Mycobacterium tuberculosis to construct a gene vaccine.Balb/c mice were muscularly injected with recombinant DNA vaccine;negative control (mice were injected with normal saline), positive control (mice were injected with DNA vaccine harboring tandem CEA epitopes plus aluminium hydroxide adjuvant) and experimental group(mice were injected with PCITri CEA_625-667-met HSP70) were set up.After 3 inoculations,the mice were sacrificed and their splenocytes and sera were collected.T cell subsets were analyzed by FCM.IFNγ in their cultural supernatants and seral IgG antibody against CEA were detected with ELISA.Results The amount of CD3⁺and CD4⁺T cells in the mouse spleen in negative control was 55.1%±6.1% and 30.2%±4.1% with no production of IFNγ by in vitro culture of spleenocytes,which could be considered as basal physiological parameters.The fused DNA vaccine induced vigorous T cell responses in mice with nearly 50-fold increases of spleen CD3⁺and CD4⁺T cells and enhanced IFNγ production up to 3 folds for non-specificity and 6 folds 　to specific challenge in cultured splenocytes under stimulation of CEA peptide (P<0.01).The serum titer of IgG antibody against CEA epitope in negative control group was 0 and less than 1∶500 in positive control,whereas that in the mice immunized with the DNA vaccine encoding the fusion protein of the CEA epitopes and HSP70 was up to 1∶4000.Conclusion DNA vaccine for the fusion protein consisted of CEA repeat epitopes and heterogenous HSP70 fragment can induce a strong immune response in mice featured by stimulation of T helper cells.

Objective To get the protein expression of sFlt-1 by transfection and to investigate the effects of sFlt-1 gene transfection on the growth of K562 cells. Methods The recombinant plasmid pcDNA₃-sFlt-1_D4 was constructed.The recombinant plasmid pcDNA3-sFlt-1_D4 was transfected into the bone marrow stromal cells by Lipofectamine 2000，which was identified by RT-PCR,ELISA and MTT.Results The transfection efficiency identified by flow cytometry was 9.27%.The protein expression of sFlt-1D4 was found in the culture supernatant 24 h,48 h and 4 weeks after transfetion by ELISA and the expression concentrations were（0.104±0.078 ），(0.158±0.022) and（0.171±0.069） μg•L^-1，respectively.The content of VEGF secreted by K562 culturing with transfectant cells culture supernatant was reduced compared with control.The inhibitoy rates on the proliferation of K562 cells via MTT assay were 9.41%±4.71%，23.63%±7.50%， and 33.13%±6.93%，respectively. Conclusion The bone marrow stromal cells transfected with recombinant plasmid pcDNA3-sFlt-1_D4 could secrete sFlt-1_D4 and inhibit the proliferation of K562 cells.

Objective To investigate the effects of indomethacin on proliferation and inducible nitric oxide synthase(iNOS) activity of laryngeal cancer cell Hep-2 to provid theoretical evidence for precaution and healing of laryngeal cancer. Methods Hep-2 cells were exposed to indomethacin at concentrations of 30，60,125,125 μmol•L^-1 and cultivated for 24 and 48 h;MTT and trypan blue exclusion experiments were used to determine cell proliferation and cell viability respectively, and compared with control group. Hep-2 cells were treated with indomethacin（125,250,500 μmol•L^-1） and lipopolysacchairde(LPS),10 μmol•L^-1) for 16 h,control group with LPS alone was set up. Enzymic method and spectrophotometric method were used to determine the activity of iNOS in cultured supernatant. Results Compared with control group,as the increase of dosage, the inhibitory rates of proliferation of cancer cell Hep-2 in different concentrations of indomethacin groups increased significantly （P<0.05）. While the identical concentrations of indomethacin were administered for 24 and 48 h, the inhibitory rates of proliferation of cancer cell Hep-2 decreased significantly（P<0.05）. Compared with control group,as the increase of dosage, the inhibition of cell viability of cancer cell Hep-2 in different concentrations of indomethacin groups decreased significantly （P<0.05）; Compared with LPS control group, indomethacin (125, 250, and 500 μmol•L^-1) decreased the LPS-induced iNOS activity significantly（P<0.05）in the supernatant. Conclusion Indomethacin, within the concentration range of 30-250 μmol•L^-1，has strong inhibitory effect on Hep-2 cell proliferation and viability and may decrease LPS-induced cell iNOS activity significantly .

Objective To observe the effects of fluvastatin on proliferation and apoptosis of HL-60 cells,and to offer the theoretical evidence for tumor treatment.Methods HL-60 cells were divided into: fluvastatin groups (0.5,5.0,10.0 and 20.0 μmol•L^-1),HL-60 control group,positive control group(treated with 10.0 μmol•L^-1ATRA).　The live cell number was counted for cell proliferation assay.The growth inhibitory rate of HL-60 cells was detected using CCK-8 kit.The cell cycle distribution and apoptotic rate were measured using flow cytometry assay.Results Compared with control group,after HL-60 cells were treated with 0.5,5.0,10.0 and 20.0 μmol•L^-1of fluvastatin for 1-4 d,the number of live cells decreased in different level(P<0.01),and in a dose- and time-dependent manner .After HL-60 cells were treated with fluvastatin for 2-72 h,the inhibitory rate of cell growth was markedly elevated,and the inhibitory rate further increased along with doses of fluvastatin and action time.The progressively increased cell growth inhibition was found in 10.0 and 20.0 μmol•L^-1 fluvastatin groups after treated for 48 and 72 h compared with the same concentration after treated for 2 h(P<0.01).Flow cytometry assays exhibited that the percentage of G₀/G₁ phase cells elevated (P<0.05),the percentages of S phase cells descended significantly,and the apoptotic rate increased after HL-60 cells were exposed to 20 μmol•L^-1 fluvastatin for 48 h,compared with control group(P<0.01).Fluvastatin-induced cell cycle distribution and apoptosis were reversed by coexposure to fluvastatin and mevalonate,Conclusion Fluvastatin inhibits proliferation and induces apoptosis of HL-60 cells,and these effects are mediated through mevalonate pathway.

Objective To investigate the effects of all-trans-retinoic acid (ATRA) on proliferation of cultured human retinal pigment epithelium (RPE) cells and the probable mechanisms. Methods Cultured human RPE cells were treated with various concentrations (10^-9,10^-8,10^-7,10^-6 and 10^-5 mol•L^-1) of ATRA at different time points (6,12,24,48,72 and 96 h).Cell proliferation was evaluated by cell count and MTT colorimetric assay,and cell cycle analysis was performed by flow cytometry. Results The cell viability rates of ATRA treated group were decreased obviously, compared with control groups (P<0.01).The inhibitory rates of RPE cell proliferation had positive correlation with ATRA dose and time of action (r₁=0.9926 ,P<0.05; r₂=0.9647,P<0.05).In ATRA groups,compared with non-ATRA treated groups,the cell number in G₁ phase was increased (P<0.05),while in G₀/G₁ phase was decreased (P<0.05).Conclusion ATRA could inhibit the proliferation of human RPE cells in a dose-dependent and time-dependent manner by arresting cell cycle.

Objective To reproduce the inflammatory bowel disease（IBD） models i nduced by 2，4，6-trinitrobenzene sulfonic acid（TNBS），and to investigate the effect of Changkangyin（CKY） on it.Methods Total　 60 rats were divided into 6 groups by random：alcohol control group，model group，salicylazosulfapyridine （SASP）group，low，middle and high dosages CKY groups.IBD model was induced by TNBS.The changes of macroscopic morphous and pathohistology of colon were observ ed after intervened with drug for 21 d. Results The body weight in model group was obviously lower than that in alcohol control group （P<0.01）;the body weights in low，middle and high dosages CKY groups and SASP group were higher than that in model group（P<0.01）;the body weight in high dosage CKY group was higher than that in SASP group（P<0.01）.The scores of macroscopic morphous and pathohistology in model group were higher than those in alcohol control group obviously （P<0.01）; 　the scores in low，middle and high dosage CKY groups and SASP group were lower than those in model group obviously（P<0.01 or P<0.05）;there was no significant difference between high dosage CKY group and SASP group（P＞0.05）.Conclusion Colitis can be induced by TNBS in rats.The change of it is the same as human’s.It is a ideal model to study pathogenesis of IBD and investigate the effect of medicine.CKY has a batter therapeutic effect on IBD model.

Objective To construct a recombinant expression vector of human inter leukin-24 (hIL-24) gene and express it in E. coli M15, and to evaluate the bioactivity of IL-24 fusion protein． Methods The human IL-24 cDNA fragment was amplified from plasmid by polymerase chain reaction(PCR),and sequenced.PQE/hIL-24 was constructed by gene rearrangement,then it was transformed into E. coli M₁ 5.The expression of the target protein was induced with IPTG and purified by Ni²⁺ -NTA agarose column.The expressed recombinant hIL-24(rhIL-24) was identified by SDS-PAGE and Western blotting. Normal peripheral blood mononuclear cells(PBMCs) were cultivated with the expression protein for 48 and 72 h，the levels of IL-6,IFN-γ and TNF-α of PBMC sstimulated with rhIL-24 were detected by ELISA． Results The recombinant prokaryotic expression vector PQE/IL-24 was constructed successfully and expressed stably in E. coli M₁5. At about 18　400 of molecular weight,there was an induced protein band.The levels of IFN-γ, IL-6 and TNF-α in the group of cultivated with the expression protein were obviously higher than those in the groups without the expresson protein(P<0.05).The rhIL-24 had a strong bioactivity with natural IL-24 protein. Conclusion The recombinant expression vector PQE-IL-24 has been constructed successfully and has efficency expression in E.coli M15，rhIL-24 which biologic activties are same as natural IL-24 protein is obtained．

Objective To construct prokaryotic expression plasmid of human recom binant soluble TNF-related apoptosis inducing ligand(rsTRAIL), then to investig ate the effects of rsTRAIL on apoptosis in non-small cell lung carcinoma（NSCLC）. Methods The encoding sequence for rsTRAIL was amplified with RT-PCR and cloned into PQE₃0 vector to establish the prokaryotic expression system. The competent cells of host strain of M₁5 were transformed by the recombinant plasmid. The expression of the target protein was induced with IPTG and purified by Ni²⁺ -NTA agarose column. rsTRAIL was added in the media of A549 and H460^wt cells, then the viability was examined by MTT assay. Apoptosis of H460^wt cells was observed under fluorescence microscopy. The apoptotic rate of tumor cells was examined by FACS. Results The cloned fragment of rsTRAIL was 100% consistent with that reported in GenBank. The expressed protein with molecular weight of 21 000 in SDS-PAGE as expected was obtained and recognized by a commercial McAb. The apoptotic rate of H460^wt cells after treated with rsTRAIL for 24 h was 43.2%. Cislatin enhanced the effect of rsTRAIL on A549 cells. Conclusion The rsTRAIL is obtained after Ni affinity chromatograph. rsTRAIL has a strong cytotoxic activity against NSCLC and cisplatin may enhance the antit umor effect of rsTRAIL .

Objective To investigate the expression of tight junctions protein claudin-6 in breast cancer cell lines and tissues and its relationship with metastasis.Methods RT-PCR and immunohistochemistry analysis were used to detect claudin-6 mRNA and protein expressions in human mammary cancer tissues and cell lines. Results claudin-6 gene was expressed in mammary gland epithelial cell HBL-100，but undetectable in other cell lines；compared with benign mammary gland,fibroadenoma，claudin-6 was down-regulated in breast cancer（P<0.05）.Furthermore，the down -regulation had significantly negative correlation with mammary tumor pathological grades and metastasis（P<0.05）,but not related to age and clinical stages （P＞0.05）.Conclusion claudin-6 may play a negative regulatory effect on mammary carcinogenesis and metastasis,and may be a potential indicator for mammary cancer metastasis and prognosis.

Abstract:Objective To research the role of Helicobacter pylori (Hp) on chronic hepatitis B.Methods The seroprevalence of Hp infection and the quantity and genotyping of HBV DNA in 376 patients with chronic hepatitis B,including chronichepatitis group,cirrhosis group and HBV-related hepatocellular carcinoma (HCC)group,were detected,and compared with control and gastritis groups. Results Hp seropositivities in chronic hepatitis B group (56.2%),cirrhosis group (69.9%),HCC group (75.0%) were higher than that in control group (43.4%) (P<0.01),and had no significant difference with chronic gastritis group (57.9%) (P>0. 05),the Hp seropositivities in cirrhosis and HCC groups were higher than that in chronic hepatitis group (P<0.05).With the different quantities of HBV,Hp seropositivities all increased compared with control group (P<0.01); but there was no significant difference between the different quantities of virus.Hp seropositivities in chronic hepatitis patients with genotype B，C and D were 61.3%,63.3%,and 50.0%,respectively,but there was no significant difference between patients with different HBV genotypes (P>0.05). Conclusion Seroprevalence of antibodies to Hp in patients with chronic hepatitis B increases significantly，and Hp seropositivity increases with the pathological changes of chronic hepatitis B.

Objective To detect T lymphocytes subset and Th1/Th2 of peripheral blood from patients with idiopathic pulmonary fibrosis(IPF) by flow cytometry (FCM) and evaluate the role of them in pathogenesis and clinical significance. Methods The levels of T lymphocyte subset (CD3⁺,CD4⁺, CD8⁺), IFN-γ belonged to Th1 and IL-4 belonged to Th2 in peripheral blood mononuclear cells(PBMC)were detected by FCM and the ratio of CD4⁺/CD8⁺ ,Th1/Th2 were analyzed from 25 patients with IPF. 25 healthy volunteers were used as controls. Results The level of CD4⁺ and the ratio of CD4⁺/ CD8⁺ in IPF group were lower than those in control group(P<0.05). The level of CD8⁺ in IPF group was obviously higher than that in control group(P<0.01),while CD3⁺ had no change (P>0.05). The level of IL-4 in IPF group was significantly higher than that in control group(P<0.01),while the level of IFN-γ had no change(P>0.05).The ratio of Th1/Th2 in IPF group was significantly lower than that in control group (P<0.01). Conclusion The excessive cell immunity and immunodominance of Th2 with disbalance of Th1/Th2 may participate in pathogenesis of IPF,which will supply rationale foundation for diagnosis and cytokine therapy of IPF.

Abstract:Objective To investigate the proportion of CD4⁺CD25^highTreg and CD4⁺CD25⁺CD127^low/-Treg in peripheral blood in patients with chronic hepatitis B (CHB) and determine the relationship between the proportion of CD4⁺CD25⁺Treg and clinical parameters. Methods Fresh isolated peripheral blood mononuclear cells (PBMCs) of 28 patients with CHB and 24 healthy donors were analyzed for the proportion of CD4⁺CD25⁺Treg using flow cytometry by surface staining for CD4-PC5，CD25-FITC，CD127-PE. HBsAg,HBsAb,HBeAg,HBeAb and HBcAb were evaluated.HBV DNA levels were measured using real-time RT-PCR. Results The proportions of CD4⁺CD25^highTreg to CD4⁺ T cells (3.36%±2.59%) and CD4⁺CD25⁺CD127^low/-Treg (4.05%±1.63%) to CD4⁺ T cells in patients with CHB were higher than those inhealth controls(1.60%±0.66%, 1.75%±0.83%, P<0.01) in peripheral blood. The CHB patients with high level alanine amino transferase (ALT) (ALT>100U•L^-1) had a higher fraction (4.26%±3.10%) of CD4⁺CD25^highTreg in peripheral blood than those patients with low level ALT (ALT＜100U•L^-1,2.52%±2.72%,P<0.05). The percentage of CD4⁺CD25^highTreg was correlated with ALT level (r=0.44，P<0.05). Conclusion The level of Treg in CHB patients is higher than that in health controls. The expression of CD25 could be influenced by the level of ALT. As a specific cell surface marker, CD127 can be helpful to obtain pure CD4⁺CD25⁺Treg.

Objective To study the expression of fragile histidine triad ( FHIT) in oral squamous cell carcinoma (OSCC),and discuss its role and significance in OSCC.Methods Immunohistochemical method was used to detect the expressions of FHIT in 48 cases of OSCC and 26 normal oral mucosa.Results The positive expression rate of FHIT in normal oral mucosa was 76.92%(20/26).Among 48 OSCC patients，the positive rate of FHIT (43.75% ) was lower than that in normal oral mucosa ( P<0.05).The expression of FHIT had no significant correlation with age and sex of patients ( P>0.05),but FHIT protein content was significantly associated with differentiation ( P<0.05),the poorer the differentiation,the less the expression.Conclusion The expression of FHIT is less in OSCC patients.FHIT plays an important inhibitory effect on the occurrence and development of OSCC.

Objective To study the expression of 17β-hydroxysteroid dehydrogenase(17β-HSD) type 2 in breast cancer and adjacent non-malignant tissue,and the correlations between the results and clinical factors.Methods The expressions of the oxidative enzyme 17β-HSD type 2 in 76 specimens of female human breast cancer and adjacent non-malignant tissues were determined by immunohistochemistry.The relationships between the clinical parameters including patient’〖KG-*3〗s age, ER,PR,P185,the stage of breast cancer,tumor size and the percentage of involved lymph nodes were analyzed.Results 17β-HSD type 2 was found in the cytoplasm of tumor cells in 5.3% of cases.In adjacent non-malignant tissues,82.9% of cases were positive and the enzyme was detected in the cytoplasm of epithelial cells of the acini and ducts.There was a significant difference between the malignant tissue and the adjacent non-malignant tissue in the percentage of positive cells(χ²=92.908,P<0.001).In adjacent non-malignant tissues,the expression of 17β-HSD type 2 was negatively correlated with the tumor size（r=－0.341，P<0.05），the stage of the breast cancer（r=－0.706，P<0.01） and was positively correlated with the patient's age（r=0.677，P<0.01）. For the other clinical parameters including ER,PR,P185 and the percentage of involved lymph nodes,there was no significant correlation.No significant correlation was found between the expression of 17β-HSD type 2 and all clinical parameters in breast cancer tissue.Conclusion 17β-HSD type 2 can regulate the local estrogen level.It may play a significant role in the development and/or progression of breast cancer.

Abstract:ObjectiveTo investigate the effect of anti-sense osteopontin （ANOPN）on proliferation and metastasis of esophagus cancer cells.MethodsAn OPN gene recombinant expression vector plasmid was constructed by RT-PCR from human umbilical vein endothelial cell gene and cloned into a mammalian expression vector pcDNA3.1(+).PcDNA3.1-ANOPN was introduced by LipofectinTM .Positive cell clones (ECA-ANOPN) ,vector-transfected cells ECA-vect and blank cell ECA were used as three groups.RT-PCR and immunocytochemistry assay were used to investigate the expressions of OPN mRNA and protein.The metastasis characteristics of cells were studied by Transwell method.Results The vector was constructed successfully,the sequencing result was identical with that reported in GenBank.Compared with vector-transfected cells (ECA-vect cells) and ECA cells，the growth rate of ECA-ANOPN cells was significantly slowed (P<0.05) ,the expression rate was decreased (P<0.05), their doubling-time increased (P<0.05),and the number of ECA-ANOPN cells in mucosa was decreased(P<0.01).Conclusion The stable expression of ANOPN gene can significantly suppress the malignant phenotype of ECA109 cells.

Objective To examine the expression of angiopoietin-2（Ang-2） in normal ovary and epithelia ovarin carcinoma,and discuss the relationship between Ang-2 and clinical pathological features of epithelia ovarian carcinoma.Methods The expressions of Ang-2 and CD34 in 46 paraffin specimens of epithelia ovarian carcinoma and 25 paraffin specimens of normal ovary were detected by immunohistochemistry SABC technique,and the microvessel density（MVD）of tissues signed by CD34 was counted. Results The expression of Ang-2 in epithelia ovarian carcinoma was higher than that in normal ovary tissue (P<0.05). The expression of Ang-2 in the stageⅠand Ⅱepithelia ovarian carcinoma was lower than that in stage Ⅲ and Ⅳ（P<0.05）;The expression of Ang-2 in epithelia ovarian carcinoma was not correlated to types and degrees of histology. There was a remarkable positive correlation between the expression of Ang-2 and MVD in epithelia ovarian carcinoma（r=0.733，P<0.01）.Conclusion The expression of Ang-2 might be related to the development of epithelia ovarian carcinoma,and may promote the development of epithelia ovarian carcinoma by promoting angiogenesis in tumor.

Abstract:Objective To explore the change of CD4⁺ CD25⁺ regulatory T cells(Treg) from peripheral blood in patiens with multiple organ dysfunction syndrome(MODS) before and after treatment and its significance.Methods The 　frequencies of CD4⁺ CD25⁺ CD127^-Treg were detected in 10 patients with MODS respectively before treatment and 1 week after treatment and 10 healthy donors by flow cytometry labeled with specific fluorescent antibodies,such as anti-CD4(PE-CY5) ,anti-CD25(FITC) and anti-CD127(PE).Results The frequency of CD4⁺ CD25⁺ CD127^-Treg in patients after treatment(2.11％±0.33％) was significantly lower than before treatment (7.44％±1.59％) (P<0.05）,and they were all higher than that of healthy donors(P<0.05).Conclusion The frequency of CD4⁺ CD25⁺ CD127^-Treg increases in patients with MODS.That may surpress the process of MODS.

Abstract:Objective To investigate the correlation between male infertility and Y chromosome microdeletions of azoospermia factor (AZF) regions.Methods Multiplex PCR amplification of 9 sequence-tagged sites in AZF regions of Y chromosome was examined among 107 infertile male patients with azoospermia(n=83) or oligozoospermia(n=24)and 20 fertile men as controls .Results Among 107 patients,the rate of Y chromosomal microdeletions was 10.3%(11/107),and the rates of azoospermia and oligozoospermia were 9.6%(8/83) and 12.5%(3/24).Among 11 patients with microdeletions of Y chromosome，5 patients had deletion of AZFc region alone,4 had deletion of AZFc and AZFd regions,1 had deletion of AZFb+c regions ,1 had deletion of AZFb+c+d regions.Moreover,8 and 11 patients had deletion of SY 254 and SY 255,respectively.No positive one was found in the controls.Conclusion Y chromosomal microdeletion is one of the major causes of severe dyszooospermia.

Objective To investigate the expression of a novel imprinted gene,PEG10,in human gastric adenocarcinoma,and the effect of PEG10 on cell growth and proliferation of gastric cancer cells.Methods The PEG10 mRNA expressions in 40 human gastric adenocarcinoma,the corresponding adjacent normal tissues and 6 nomal gastric tissues were detected by RT-PCR.The expression vectors of PEG10 were constructed and transfected into gastric cancer cell line MKN45 which had no endogenetic PEG10 expression.Cell growth ability was measured by MTT assay.Results High PEG10 mRNA expression level was detected in 9 of 20(45.0%) human gastric adenocarcinoma which was significantly higher than those of the matched normal tissues(10.0%) (Ｐ<0.05).　No expression of PEG10 in nomal gastric tissues and gastric cancer cell line MKN45.The growth ability of transfected MKN45 cells was increased.Conclusion The imprinted gene PEG10 can overexpress in human gastric adenocarcinoma and enhance the growth ability of gastric cancer cells.

Abstract:Objective To investigate the expression patterns of EphB4 in non-small cell lung cancer(NSCLC) and evaluate their roles in tumor initiation and development.Methods The expressions of EphB4 were detected with immunohistochemical method in 34 cases of NSCLC and 16 cases of adjacent normal lung tissues.Results The positive expression rate of EphB4 in NSCLC was 41.2%（14/34）.The expression of EphB4 in 16 cases of adjacent normal lung tissue were all negative,there was significant difference between them(P<0.01).The positive expression of EphB4 was closely related to differentiation and clinical stage(P<0.05),but not to histological classification,age,sex and lymph node metastasis (P>0.05).Conclusion EphB4 may play a key role in carcinogenesis and progression of NSCLC.