To observe the enhancing effect and reducing toxicity of 20(S)-protopanaxadiol (PPD) in combination with cyclophosphamide (CTX) on transplanated tumor human lung carcinoma A549 cells.Methods The transplanted tumor models with human lung carcinoma A549 cells were established and then divided randomly into six groups: control group,PPD group,low dose CTX group,high dose CTX group,PPD combined with low dose CTX group and PPD combined with high dose CTX group.The mice in each group were injected intraperitoneally with same amount of CMC,PPD (50 mg•kg^-1•d^-1),CTX (10 mg•kg^-1•d^-1),〖JP2〗CTX (20 mg•kg^-1•d^-1),CTX(10 mg•kg^-1•d^-1)+PPD (50 mg•kg^-1•d^-1) and PPD (50 mg•kg^-1•d^-1)+ CTX (20 mg•kg^-1•d^-1),respectively.After administration for 15 d,the mice were killed and the mouse weight,tumor weight and volume,the number of WBC in peripheral blood, the count of bone marrow nucleated cells,spleen and thymus indexes,proliferation of T lymphocytes and nature killer cell activity were detected.At the same time,the apoptotic rate and activity of caspase-3 were analyzed by flow cytometry.Results Compared with CTX groups,the inhibitory rate of tumor in PPD(50 mg•kg^-1) +CTX group increased (P<0.01),the count of bone marrow nucleated cells increased (P<0.01),the spleen and thymus indexes increased (P<0.01), the proliferation ability of T lymphocytes (P<0.05)and nature killer cell activity increased (P<0.01). Compared with CTX groups,the apoptotic rates(P<0.01) and activities of caspase-3 in PPD+CTX groups increased significantly(P<0.05),but there was no significant difference between PPD and CTX groups.Conclusion PPD could significantly enhance the anti-tumor activity of CTX against lung cancer on nude mice and reduce the side effects simultaneously by up-regulating the activity of caspase-3 and inducing the apoptosis.

Abstract:Objective To construct the eukaryotic expression recombinant PIAS3 plasmid fused with Myc protein and express the fusion protein Myc-PIAS3.Methods The full length PIAS3 fragment of 1 851bp was amplified by PCR and ligated into pMD18-T vector. The full length PIAS3 fragment was subcloned into eukaryotic pCMV-Myc vector between SalⅠ and NotⅠ sites.The recombinant pCMV-Myc-PIAS3 plasmid was trandfected into PC3 cells.The eukaryotic expression Myc-PIAS3 protein was checked by Western blotting with Myc antibody.Results The recombinant plasmid showed right sequence by the full length sequencing.The recombinant plasmid of pCMV-Myc-PIAS3 was identified by enzyme digestion.As expected,by EcoRⅠ digestion,it showed two bands of 4 357bp and 1 318 bp. By XbaⅠdigestion,it showed two bands of 3 291 bp and 2 384 bp.The sequencing result showed a N-terminal Myc of 13 amino acids followed with PIAS3 gene sequence in right reading frame.The pCMV-Myc-PIAS3 plasmid was transfected into prostate cancer PC3 cells.A specific protein expression band at relative molecular mass 68 000 was obtained by using Myc-antibody with Western blotting method.Conclusion The recombinant plasmid of pCMV-Myc-PIAS3 is sucssesefully constructed,and Myc-PIAS3 fusion protein is sucssesefully expressed.

Abstract:Objective To study the preventive effect of curcumin (Cur) on ultraviolet B (UVB) oxidation carcinogenesis in HaCaT cells and its mechanisms. Methods 1.25,2.50 and 5.00 mg•L^-1 Cur were added into HaCaT cells irradiated by UVB and the cells were cultivated continually.The contents of oxides and antioxides were determined with zymochemistry method;the Δψ potential and the ［Ca²⁺］i were measured with Rhodamine 123 and Fluo-3/AM fluorescence.The protein expressions of apoptotic correlating factors were examined by flow cytometry. Results Compared with control group,the Δψ of the HaCaT cells ,the contents of SOD and GSH-Px and the expression of Bcl-2 protein in UVB irradiation groups decreased significantly after UVB irradiation (P<0.01);while the ［Ca²⁺］i,the contents of ROS,NO and MDA,and the protein expressions of caspase-3,cytochrome c and Bax increased significantly (P<0.01). After Cur was added into the cells irradiated with UVB , the Δψ and the expression of Bcl-2 in every Cur group increased significantly,the contents of SOD and GSH-Px in 2.50 and 5.00 mg•L^-1 Cur groups increased significantly(P<0.01),while the contents of ROS,NO and MDA and the protein expressions of caspase-3,cytochrom c and Bax in every Cur group and the ［Ca²⁺］i in 2.50 and 5.00 mg•L^-1 Cur groups decreased significantly compared with UVB irradiation group(P<0.01).The ratio of Bcl-2/Bax varied within 0.58-2.50 and Cur showed a pattern of dose-effect. Conclusion Cur could increase the Δψ and the antioxidase activity and decrease the ［Ca²⁺］i,the contents of oxides and the protein expressions of induced apoptotic correlating factors. Cur has preventive effect on cancinogenesis of the cells irradiated by UVB.

Abstract:Objective To investigate the effects and mechanism of betulin on rats with alcoholic liver disease. Methods Rats were randomly divided into six groups: normal group,alcoholic liver disease(ALD) model group,betulin low-dose group,betulin middle-dose group,betulin high-dose group and positive control group (silbinin).The ALD model rats were made by feeding with alcohol,the rats in positive control group were administered with silbinin(100 mg•kg^-1•d^-1),the rats in low-dose group,middle-dose group and high-dose group were administered with betulin(10,20 and 40 mg•kg^-1•d^-1 respectively).The activities of ALT,AST,ALP,GGT in serum,the level of TG,CHO,HDL,LDL in serum and TG in liver tissue were measured after administration for 15 d.HE staining was used to observe the pathological changes in liver. Results Compared with normal control group, the activities of serum ALT,AST,ALP and GGT and the levels of serum TG,CHO and LDL in ALD model group increased significantly.HDL decreased significantly.The TG level in liver tissue increased significantly.Liver tissue was damaged seriously,hepatic lobule structure disappeared,cells swelled balloon-like change.Various sizes and number of lipid droplets were seen in cytoplasm of liver cells swelled.Compared with ALD model group,high-dose and middle-dose of betulin significantly decreased the activities of ALT,AST,ALP,GGT in serum, the levels of TG and CHO in serum and the TG level in liver tissue (P<0.01).The hepatic injuries of rats in high-dose betulin group were recovered obviously.Conclusion High-dose of betulin has obviously protective effects on alcoholic liver injury in rats,the mechanisms probably contribute to reducing lipid peroxide and reducing fatty sediment in liver.

Abstract:Objective To study the effect of Hypocretin neuropeptide in hypothalamic nuclei participating in regulation of habenular nucleus(Hb) on sleep-wakeness process and to provid theoretical basis for it. Methods Zolpidem　 (5 mg•kg^-1) and saline were administered respectively to adult wistar rats by gavage in experimental group(n=10) and control group(n=10) and the Hb tissue was extracted when the drug action was at peak.The expression of HcrtR2 mRNA of Hb was determined by using RT-PCR technique. Choleratoxin B solution was microinjected into the isolateral Hb of rats (n=5) to retrograde and observe whether labelled cells existed in hypothalumic nuclei.Results The expression of HcrtR2 mRNA in the Hb of experimental group（0.13±0.03） was lower than that of control group (0.26±0.04,P<0.05).The choleratoxin B labelled cells distributed in the lateral hypothalamic area,the lateral preoptic area and the arcuate nucleus. Conclusion Hypocretin neuronpeptide in hypothalamic nuclei and its receptor are involved in the regulation of the Hb on sleep-wakeness process.The Hb is innerved by the lateral hypothalamic area,the lateral preoptic area and the arcuate nucleus.

Abstract:Objective To establish the culture methods of submandibular gland cells from Wistar rats in vitro and lay a foundation for amplification of submandibular gland cells and gene therapy of salivary diseases.Methods The submandibular gland was obtained from Wistar rats which were one day old,after digested with LB3 and pancreatin，the submandibular gland cells were cultivated in DMEM/F12 culture fluid containing 2% fetal calf serum,epidermal gowth factor and insulin.The morphology of the culture and subculture cells was observed by phase contract microscope.The cell origin was identified by immunohistostaining.PAS staining was used to observe cell function of synthesis and secretion of polysaccharides.Results The morphous of primary cultured cells was round,triangle or polygon.At passage 2,the cells grew well,there was no change of morphous.The cells were positively stained with cytokeratin,E- cadherin and PAS,it indicated that the cells were epithelium origin and had the function of synthesis and secretion of polysaccharides.Conclusion The Wistar rat submandibular gland cells are successfully isolated and cultivated with enzyme digestion.The cultured submandibular gland cells at passage 2 still maintain their biocharacteristics.

Abstract:Objective To observe the protective effects of Jiangzhitong on carbon tetrachloride-induced chronic hepatic injury in rats and find its possible mechanisms. Methods Seventy two rats were randomly divided into 6 groups: control,model,positive control and jiangzhitong groups with different doses (50,100，200 mg•kg^-1•d^-1).　The effects of Jiangzhitong on hepatic injury were observed by measuring enzyme level (AST and ALT) in serum,reduced glutathione hormone(GSH) content and catalase(CAT) activity of hepatic tissue section. Results Compared with model group,the serum ALT and AST concentrations in Jiangzhitong groups with different doses were decreased significantly (P<0.05 or P<0.01),the GSH contents of hepatic tissue in Jiangzhitong groups with different doses were increased (P<0.05 or P<0.01).CAT activities were increased (P<0.05),Compared with positive control,the ALT concentration in high dose group was reduced obviously (P<0.01). There was no difference of ALT actvity between low,middle dose groups and control group(P>0.05).The ALT activity in high dose group was lower than those in low and middle dose groups,there was no difference of ALT activity between low and middle dose groups(P>0.05),the activities in middle and high doses groups were significantly lower than that in low dose group(P<0.05). Conclusion Jiangzhitong has the hepatoprotective effect on carbon tetrachloride-induced chronic hepatic injury in rats, its mechanism is likely related to potent antioxidative.

Abstract:Objective To investigate the hydrolysis of aconitum alkaloids in Fuzi during the process of decoction under common conditions and microwave conditions and study the changes in contents of aconitum alkaloids when Fuzi was co-decocted with different other herbs.ethods The HPLC method was used to analyze mesaconitine (MA),aconitine (AC),hypaconitine (HA) of Fuzi and their hydrolysates.Results At 30 min after heated in water by boiling,MA decreased to 10.5%,HA decreased to 41.9% and AC could not be detected.At 150 s after heated under microwave radiation,MA decreased to 59.2%,AC decreased to 41.4% and HA decreased to 86.6%;when co-decocted with rhubarb,dried ginger,liquorice, ginseng,and Radix paeoniae alba,the total contents of three diester-diterpenoid alkaloids decreased to 52.8%,66.2%,53.4%,79.5% and 83.7% of the total contents of aconitum alkaloids of Fuzi decocted alone respectively. Conclusion The hydrolysis of aconitum alkaloids of Fuzi is different in the processes of boiling under common conditions and microwave conditions;the contents of aconitum alkaloids decrease when Fuzi is co-decocted with different herbs.

Abstract:Objective To study the method of isolation and cultivation of neural stem cells from newborn rat spinal cords and identification of its differentiation，to establish cytological foundation for vicariousness therapy of peripheral nerveous system diseases by using spinal stem cells. Methods The neural stem cells were isolated by injector perfusion method from spinal cords of neonatal Wistar rat within 24 h， and induced by serum，the morphological method was used to observe the growth status of cells,anti- Nestin antibody,anti-GFAP antibody and anti-MAP-2 antibody were used for immunocytochemistry test to identify cell differentiation. Results The status of neural stem cells from newborn rat spinal cords was adherence culture in vitro，and the cells could be induced by serum to diffentiate into neural cells with typical morphology and expressing tissue-specific protein Nestin，GFAP，MAP-2. Conclusion The neural stem cells can be isolated from newborn rat spinal cords with adherence proliferation status and they have potential of differentiation.

Abstract:Objective To study the role of NF-kappaB( NF-κB) on islet cell apoptosis in high concentration glucose in vitro.Methods Pancreatic islet cells wereisolated from Kunming mice and these cells were cultivated in medium with different concerntrations of glucose: G1 (5.6 mmol•L^-1),G2(7.8 mmol•L^-1),G3(11.1 mmol•L^-1),G4(16.7 mmol•L^-1),G5(22.2 mmol•L^-1),G6(27.6 mmol•L^-1).After the slet cells were cultivated for 72 h,insulin secretion was evaluated by radio-immunity technique.The NF-κB expression was detected by immunocytochemistry and cytochrome C(Cyt.C) release was detected by immunofluorescence technique.Apoptosis of islet cells was determined by Hoechst33342 assay. Results Insulin secretion was enhanced and NF-κB activity was increased（P<0.05）when the concentrations of glucose were 5.6-11.1 mmol•L^-1,but Cyt.C release and apoptosis didn’t change. When the concentration of glucose exceeded 16.7 mmol•L^-1,the apoptotic percentages of islet cells were increased signficantly accompanying with the glucose concentration elevation compared with G₁,G₂ and G₃ groups(P<0.05),the NF-κB activity and Cyt.C relesae were significantly increased （P<0.05）.Conclusion High glucose can induce the apoptosis of islet cells and decrease insulin secretion,enhance the activity of NF-κB and Cyt C release.It suggests that the activation of NF-κB may be related to apoptosis of islet cells,and the inhibition of NF-κB may protect islet cells.

Abstract:Objective To explore the effect of sex hormone on genes related to rennin-angiotensin system(RAS) in cortex of androgen-induced sterile rats(ASR) with middle cerebral artery occlusion(MCAO). Methods The androgen-induced sterile rat model was first made,then administered with recombinant human follitropin （rhFSH）.The MCAO model was also established after rhFSH treatment.The rats were divided into four group:ASR+sham,ASR+ rhFSH+sham,ASR+MCAO,ASR+rhFSH+MCAO.There were 12 rats in each group.The brain ischemia area was measured by TTC staining. The AGT,ACE,AT1 and AT2 mRNA expressions in cortex were assayed by RT-PCR. Results TTC staining result showed that the pale rat brain ischemia areas were detected in ASR+MCAO and ASR+rhFSH+MCAO groups.The ischemia areas in ASR+MCAO group and ASR+rhFSH+MCAO group were larger than those in ASR+sham group and ASR+rhFSH+sham group （P<0.05）.The ischemia area in ASR+rhFSH+ MCAO group was smaller than that in ASR+MCAO group(P<0.05).There were no differences in AGT,ACE,AT1 and AT2 mRNA expressions between ASR+sham and ASR+rhFSH+sham groups（P>0.05）.The ACE,AT1 and AT2 mRNA expressions in ischemia side in ASR+MCAO group were obviously higher than those in ASR+sham group（P<0.05）,but the AGT mRNA did not change（P>0.05）.There were no differences in AGT,ACE,AT1 and AT2 mRNA expressions in ischemia side between ASR+rhFSH +MCAO group and ASR+rhFSH+sham group（P>0.05）.Conclusion The activation of RAS induced by androgen increasing is involved in brain ischemia injury.rhFSH can lessen the brain ischemia injury by down-regulating RAS.

Abstract:Objective To detect the characterization and non cellular ROS activities of nano silica particle and provide references for predicting its toxicity in vivo and in vitro.Methods Transmission electron microscope(TEM) was used to observe particle size,shape and dispersibility of the two silica particles;the size and agglomeration state of particles in water,saline,10% Tween 80,RPMI 1640 and RPMI1640 containing 1% FBS were measured in 0,24,48 and 72 h after 30 s-sonication by Zeta Potential Analyzer;non cellular ROS activity was detected using DCFH-DA regents.Results TEM image showed nano silica particles were round and dispersed evenly;the average sizes of the two silica particles were (43.0±4.2) and (68.0±5.7) nm.Silica particles had different sizes in water,saline,10% Tween 80,RPMI 1640 and RPMI1 640 containing 1% FBS,among them,two particles had smallest sizes in saline,which were 74.0 and 96.7 nm,respectively.And at 0,24,48 and 72 h after 30 s-sonication,Si-43 nm and Si-68 nm did not form agglomeration in water but formed similar agglomeration in RPMI 1640 cell culture medium containing 1% FBS.The two nanoparticles hardly caused any increasing of ROS between 30-100 μg mass range in cell free system,just equivalent 2.5 μmol•L^-1 H₂O₂. Conclusion Nano silica particles could form agglomerations in cell culture medium and almost have no ROS activities in non cellular system.

Abstract:Objective To identify the effect of morphology of adult upper and middle thoracic pedicle on security of screw implantation.Methods Five fresh adult cadaveric thoracic spine from T1 to T8 were harvested.Pedicle transverse diameters were measured and compared with the smallest diameter(4.5 mm) of screw used in clinic.The screws were inserted and implant effect was observed.Results The mean transverse diameter of adult thoracic（T1-T8） were T1:4.74（3.85-5.25）mm,T2:4.51（3.45-5.25）mm,T3:4.15（3.60-4.65）mm,T4:3.75（3.10-4.50）mm,T5:4.25（3.70-4.65）mm,T6: 4.53（4.40-4.70）mm,T7:5.38（5.25-5.60）mm,T8:5.55（5.50-5.80）mm.Of all specimens,there were 9 specmen(22.5%) whose tranverse diameters were smaller than 4.5 mm.After implantation,two screws broke through pedicle lateral wall. Conclusion Adult pedicle transverse diameters in T3-T4 are smallest,transpedicular screw placement is difficult in some of these vertebrae,but in most of thoracic vertebrae,pedicle tranverse diameter could permit screw implanting in security.

Abstract:Objective To observe H₂O₂-induced myocardial H9c2 cell apoptosis and protective effect of inhibitor of calcineurin (CaN) and provide experimental basis for study on cardiocytes damage caused by ischemia/reperfusion and related mechanism. Methods Myocardial H9c2 cells cultivated in vitro were treated with saline and various concentrations H₂O₂ (50,100,200,400 μmol•L^-1) at different time (1,6,24 h) in control and experimental groups respectively.Rhodamine 123 fluorescent probe was used to determine membrane potential (Δψmt).Annexin-Ⅴ/PI was used to detect myocardial H9c2 cell apoptosis.The myocardial cell apoptosis models of rats were established,myocardial H9c2 cells was treated with 100 μmol•L^-1 H₂O₂ for 6 h,FK506 were administerted to the model group at low dose (0.15 μmol•L^-1)and high dose (0.60 μmol•L^-1 )levels,and the apoptotic and necrotic rates were detected by flow cytometry. Results ① Compared with control group,H₂O₂ caused the significant increase of Δψmt in 100,200,400 μmol•L^-1 groups at 1 h（P<0.01）; Δψmt showed the significant decrease in 200 and 400 μmol•L^-1 H₂O₂ treated groups compared with 100 μmol•L^-1 group and it was significantly lower in 400 μmol•L^-1 group than in 200 μmol•L^-1 group at 6 and 24 h (P<0.05). ② Compared with control group,the apoptotic and necrotic rates were significantly increased in 200 and 400 μmol•L^-1 H₂O₂ treated groups at 1 h,and the highest in 400 μmol•L^-1 group at 24 h.③Compared with 100 μmol•L^-1 H₂O₂ treated group,the H₂O₂-induced apoptotic rate was significantly decreased in FK506 treated groups (P<0.01);there was no significant difference between low dose and high dose FK506 groups (P>0.05). Conclusion H₂O₂ could damage mitochondria,decrease mitochondrial membrane potential and induce the apoptosis and necrosis in myocardial H9c2 cells.The inhibitor of CaN shows protective effect on myocardial H9c2 cell apoptosis induced by H₂O₂.

Abstract:Objective To study the effects of chloroquine on proliferation and cell cycle of hepatocellular carcinoma cell line HepG2 cultured in vitro　and provide basis for research on chloroquine in therapy for liver cancer.Methods HepG2 cells were divided into six groups：control group and chloroquine (8.00，16.00，32.00，64.00 and 128.00 mmol•L^-1 )groups.The cell viability was determined by MTT,the cell cycle and apoptosis were detected by flow cytometry.Results Compared with control group，the cell viabilities were inhibited significantly 24 h after treated with chloroquine (32.00-128.00 mmol•L^-1) or 48-72 h after treated with chloroquine (8.00-128.00 mmol•L^-1) 　( P< 0.01) and the most obvious inhibitory effect occurred when HepG2 cells were treated with 128.00 mmol•L^-1　 chloroquine for 72 h(P< 0.01); HepG2 cells treated with chloroquine (32.00-128.00 mmol•L^-1) for 24 h were obviously arrested at G₂/M phase,compared with control group the percentage of cells at G₂/M phase and the apoptotic rate increased with the dose of chloroquine(P<0.01). Conclusion Chloroquine can suppress the activity of hepatoma HepG2 cells by inhibiting cell proliferation,inducing apoptosis and arresting cell cycle in vitro.So chloroquine may be used as the drug for treatment of hepatocellular carcinoma.

Abstract:Objective To construct eukaryotic expressing vector of human melanin-concentrating hormone receptor 2 (MCHR2)/ enhancer green fluorescent protein(EGFP) and establish a SH-SY5Y cell line expressing MCHR2 stably. Methods Human MCHR2 cDNA was amplified from the human fetal brain cDNA library by PCR. It was cloned into pEGFPN1 and the eukaryotic expressing vector pEGFPN1-MCHR2 was constructed.Then the vector was transfected into human neuroblastoma cells SH-SY5 Y by Lipofectamine^TM. After screening culture by G418,the SH-SY5Y cells expressing MCHR2 stably were established.The transcription and expression of MCHR2 were identified by RT-PCR and Western blotting．The location of MCHR2 was observed by laser confocal microscope.Results Human MCHR2 cDNA was amplified and the eukaryotic expression vector pEGFPN1-MCHR2 was constructed successfully.The expression of MCHR2 was detected by RT-PCR and Western blotting successfully.The fusion protein MCHR2/EGFP was located in the cytomembrane of the MCHR2-SH-SY5Y cells transfected with pEGFPN1-MCHR2 while EGFP was located in the cytoplasm of the SH-SY5Y cells transfected with pEGFPN1.Conclusion The cell line MCHR2-SH-SY5Y expressing MCHR2 stably has been established successfully.

Abstract:Objective To observe the migration,colonization and repairing effects of marrow mesenchymal stem cells (MSCs) on thymus tissue injury induced by ionizing radiation in mice.Methods MSCs of C57BL/6 mice were isolated,purified and cultivated in vitro.Their migration and colorization were observed with laser confocal microscopy 1,5 and 10 d after DAPI labeled. MSCs were injected into the thymus tissue of mice through tail vein.The model of thymus tissue injury induced by whole-body X-irradiation was established.The mice were divided into four groups: normal,irradiation,irradiation+saline,and irradiation+MSCs groups.The apoptosis was detected by flow cytometry and the repairing effect of MSCs on thymus tissue injury was observed by histological method 3 months later.Results The occurrence of MSCs in the thymus was observed 1 d after MSCs injection,the diffusion of MSCs in the thymus appeared 5 d later,and widely dispersed 10 d later.The apoptotic rate of thymocytes in irradiation group was higher than that in normal(P<0.05) and was lower than that in MSCs group(P<0.05).The structures of cortex and medulla of thymus were clear in mice in normal group,there were a large number of lymphocytes in the cortex and small number of lymphocytes in the medulla.The structures of cortex and medulla of thymus were unclear in mice in both irradiation,irradiation and saline groups.The lymphocytes in thymus showed extensive coagulation necrosis.There were remnants or newborn lymphoid tissue in the cortex and medulla in mice in irradiation+MSCs groups. Conclusion MSCs can be rapidly enriched in thymus tissue and promote regeneration and repair of damaged thymus.

Abstract:Objective To explore the role of mammalian target of rapamycin(mTOR) in the Hela cells injury induced by vitamin K（Vk3）through observation of mTOR,p70S6K-α1,p70S6K-α2,p70S6K-β1,p70S6K-β2 as well as cyclin D1 mRNA expression and the effects of N-acetyl cysteine(NAC) on mTOR,p70S6K-α1,p70S6K-α2,p70S6K-β1,p70S6K-β2 as well as cyclin D1 mRNA.Methods Hela cells were randomly divided into control group,Vk3 group,Vk3+NAC group and NAC group, respectively.The survival rate of Hela cells was determined by MTT.The mTOR,p70S6K-α1,p70S6K-α2,p70S6K-β1,p70S6K-β2 and cyclin D1 mRNA expressions in Hela cells were assayed by using RT-PCR.Results Compared with control group,the survival rate of Hela cells in Vk3 group was decreased（P<0.01）,the expressions of mTOR,p70S6K-α1,p70S6K-α2,p70S6K-β1,p70S6K-β2 and cyclin D1 mRNA were decreased （P<0.05）.Compared with Vk3 group,the survival rate in Vk3+NAC group was increased（P<0.01）,and the mRNA expressions of those genes mentioned above were also increased（P<0.05）.Conclusion Vk3 can inhibit Hela cell proliferation through oxidative stress,which might be correlated with down-regulation of the genes expressions of mTOR signal pathway and cyclin D1.

Abstract:Objective To investigate the protein expression in mature dendritic cells（mDCs） after transfection with total RNA from carcinoembryonic antigen(CEA)-expressing colon adenocarcinoma SW480 by electroporation and test whether electroporation can influence on the function and phenotype of DCs in order to lay a foundation for the preparation and clinical application of DCs nucleic acid tumor vaccine.Methods DCs were generated in vitro from human peripheral blood mononuclear cells(PBMC) in the presence of recombinaht human granulocyte monocyte colony stimulating factor(rhGM-CSF) and recombinant human interleukin-4 (rhIL-4),and mDCs were induced with 0tumor necrosis factor-α (TNF-α) after treated for 24 h.There were two groups in the experiment:mDCs group and immature DCs(imDCs) group.The total tumor RNA was extracted from SW480 by Trizol and was transfected into mDCs by electroporation.The phenotype of DCs and expression of CEA protein before and after electroporation were assessed by flow cytometry.The secretion of IL-12 in each group was investigated by ELISA.Results DCs with characteristic of morphological features were generated in vitro from PBMC by rhGM-CSF,rhIL-4 and TNF-α.After cultivated for 5 d，the cells gained the phenotype of imDCs,showed strong expression of CD1a,weak expression of CD80 and no expression of CD83.After maturation by TNF-α， the expressions of CD1α,CD80 and CD83 were positive and they were also positive in RNA transfection DCs group.The expression of CEA protein was meaured at 4 h after electroporation.The secretion levels of IL-12 in mDCs and RNA transfection DCs groups were significantly higher than that in imDCs group (P<0.01),but there was no significant difference between RNA transfection DCs group and mDCs group(P>0.05). Conclusion The total RNA from colon adenocarcinoma cells can be transfected i nto mDCs by electroporation and electroporation doesn’[KG-*3]t alter the phenotyp e and function of mDCs．

Abstract:Objective To investigate the protective effect of Schizandrae Lignanoid (SCL) on myocardial ischemia reperfusion injury in hyperlipidemic rats and its mechanisms and provide theoretical basis and experiment foundation for treatment of myocardial ischemia complicated with hyperlipidemic disease with SCL in the future. Methods After hyperlipidemic rat models were set upby oral administration of high lipid emulsion,the rats were divided into seven groups: control group，sham group，model group,SCL 60，20，5 mg•kg^-1 groups and 200 mg•kg^-1 CDT group.After reperfusion,the blood samples taken from the ventricles were assayed for blood lipid;MPO activities of ischemic myocardium were measured;Infarct area of left ventricles was measured by Evens blue-TTC staining,and myocardial pathological changes were also observed.Results Compared with model group, the myocardial infarct area/ventricle area ratios and MPO activities in SCL 60，20，5 mg•kg^-1 groups decreased (P<0.01) with the raise of SCL dose;In SCL 60 and 20 mg•kg^-1 groups myocardial fiber rupture and necrosis in ischemic area with less focal hemorrhage and inflammatory cell infiltration decreased;In SCL 60 and 20 mg•kg^-1 groups the levels of serum TC,TG,LDL-c,ApoB decreased and HDL-c,ApoA1 increased (P<0.05 or P<0.01）.Conclusion SCL has protective effects on myocardial ischemia reperfusion injury in hyperlipidemic rats and the mechanisms involved may be related to its regulation of blood lipid and inhibition of neutrophil infiltration.

Abstract:Objective To study the expression of prostasin in ovarian serous cystadenocarcinoma tissues and disscuss the significances of prostasin gene in the occurrence and development of ovarican carcinoma.Methods RT-PCR method was used to investigate the expressions of prostasin mRNA in tissue samples of 10 ovarian serous cystadenocarcinoma,10 ovarian serous cystadenoma and 10 normal ovary.The expressions of prostasin protein in 60 ovarian serous cystadenocarcinoma,24 ovarian serous cystadenoma and 24 normol ovarian tissues were detected by immunohistochemistry. Results The level of prostasin mRNA expression in ovarian serous cystadenocarcinoma tissues（0.415±0.177）was significantly higher than those in normal ovarian tissues（0.192±0.128）and ovarian serous cystadenoma tissues（0.237±0.232）（P<0.05）.The expression of prostasin protein was mostly located in cytoplasm;the positive expression rate of prostasin protein in ovarian serous cystadenocarcinoma group was 65.0%,which was significantly higher than those in normal ovarian group(12.5%)and ovarian serous cystadenoma group(25.0%) (P<0.01);there was correlation between the expression intensity of prostasin protein and pathological grading and clinical stages;the higher clinical stage and the worse differentiation of ovarian serous cystadenocarcinoma,the stronger positive expression of prostasin protein. Conclusion Prostasin gene plays a promotion effect on the course of occurrence and development of ovarian carcinoma.

Abstract:Objective To investigate the influence of transforming growth factor-β1(TGF-β1) on macrophage scavenger receptor (ScR) class A and B(ScR-B,CD36) in order to provide theoretical foundation for expounding the formation and therapy of atherosclerosis(AS).Methods THP-1 derived macrophages were divided into control group and experimental group,the cells in experimental group were treated with 3.0 mg•L^-1Anti TGF-β1 Ab,the cells in control group were treated with 3.0 mg•L^-1 IgG. The ¹²⁵I-acetylated low density lipoprotein (¹²⁵I-Ac-LDL for ScR-A)　 and ¹²⁵I-oxidized low density lipoprotein (¹²⁵I-Ox-LDL for CD36) uptaking (including 4℃ binding,37℃ association and degradation) were measured respectively.Influence of anti TGF-β1 antibody (Anti TGF-β1 Ab) on the ScR-A and CD36 mRNA expressions in THP-1 derived macrophages was measured meanwhile,respectively. Results Compared with control group ( binding: 8.23 μg•g^-1 ±1.24 μg•g^-1 protein,association:45.69 μg•g^-1 ±6.92 μg•g^-1 protein,and degradation: 112.18 μg•g^-1 ±20.15 μg•g^-1 protein),Anti TGF-β1 Ab increased 125I-Ac-LDL binding(48.67 μg•g^-1 ±6.52 μg•g^-1 protein),association (412.30 μg•g^-1±12.21 μg•g^-1 protein),and degradation (896.48 μg•g^-1 ±32.74 μg•g^-1 protein) significantly (P<0.01);Compared with control group (binding: 78.56 μg•g^-1 ±2.81 μg•g^-1 protein,association: 123.94 μg•g^-1 ±12.11 μg•g^-1 protein,and degradation: 345.38 μg•g^-1 ±27.17 μg•g^-1 protein),Anti TGF-β1 Ab markedly increased ¹²⁵I-Ac-LDL binding(102.32 μg•g^-1 ±3.11 μg•g^-1 protein),association (412.94 μg•g^-1±15.21 μg•g^-1 protein),and degradation (788.94 μg•g^-1 ±31.16 μg•g^-1 protein),respectively (P<0.01).The ScR-A and CD36 mRNA expressions of THP-1 derived macrophages were increased after treated with Anti TGF-β1 Ab compared with control group.The ratio of ScR-A mRNA of experimental group to control group was 1.7,the ratio of CD36 mRNA was 1.12.Conclusion TGF-β1 can intervene the formation and development of AS through inhibiting the expressions of ScR-A and CD36,and this depressive effect may be mainly depend on ScR-A.

Abstract:Objective To explore the expression of beta 2 adrenergic receptors(β₂-AR) in bone marrow cells and its significance and provide a scientific basis for the theory of hematopoietic regulation by nerve factors. Methods After　 bone marrow mononuclear cells were isolated from normal mouse bone marrow by the Ficoll density gradient centrifugation，the expressions of β₂-AR were studied by immunohistochemistry in bone marrow mononuclear cells,cultured bone marrow stromal cells,GM-CFUs,paraffin sections and smears of bone marrow.Mouse models of immune-mediated aplastic anemia(AA) were made by lymphocyte infusion.Mice were divided into 3 groups including animal model group,β₂-AR antagonist (ICI 118551) group,and control group,and the expression of β₂-AR in bone marrow mononuclear cells at the 8 th day was dete cted by Western blotting.Results The　 positive expression of β₂-AR in cytoplasm or membrane of normal mouse bone marrow cells showed different brown by immunohistochemical staining.There were intensive positive expressions of β₂-AR in mononuclear cells(48%) and bone marrow stromal cells (68%),while progenitor cells only showed middle-positive(55%)or weak positive(45%),and other hematopoietic accessory cells were positive in varying degrees.The relative values of β₂-AR of bone marrow mononuclear cells in 3 groups were 1.03±0.31,1.72±0.29 and 1.41±0.35,respectively;the expression of β₂-AR in animal model group was more lower than that in control group（P<0.05）,and the expression of β₂-AR in β₂-AR antagonist group was more higher than that in control group（P<0.05）. Conclusion β₂-AR can express in bone marrow cells of normal mice, and the expression level of β₂-AR is related to hematopoietic function of bone marrow,it may be involved in hematopoietic regulation by neuro-endocrine-immunity net work.

Abstract:Objective To investigate the expressions of activin A (Act A) and its binding protein follistatin(FS) in mice with pancreatic injury and lay a fundation for tissue repair and reestablishment after pancreas injury.Methods Twenty　 one male ICR mices were randomly divided into two groups :experimental group（n＝15）and control group（n＝ 6）,Mice were treated intraperitoneally with streptozotocin (STZ) to induce pancreatic injury.The expressions of Act A and FS protein were detected by immunohistochemistry,and the expressions of activin A and follistatin mRNA were examined by RT-PCR.Results In normal mice,Act A protein mainly distributed in islet cells,and also distributed in pancreatic acinar cells;FS protein only expressed in islet cells and not in acinar cells.Act A and FS mRNA expressed in pancreatic tissues.Compared with control group,the expression of Act A mRNA in mice in experimental group decreased gradually（P<0.01）,the expression of FS mRNA increased at the 7th day after injury,then decreased gradually at the 14th and 21st day （P<0.01）. Conclusion Act A and its binding protein FS mainly express in mouse islet tissues.The unbalance of activin Act-FS system in streptozotocin-induced pancreatic injury may be involved in the pathogenesis of diabetes mellitus.

Abstract:Objective To investigate the relation between polymorphism of c-myc and k-ras gene and thymic lymphomas of mice induced by ionizing radiation. Methods The thymic lymphomas models of mice were made by ⁶⁰Co(dose rate:0.382 Gy•min^-1， total dose: 7 Gy)exposuring to C57BL/6J and BALB/c,6 months later,the mice were killed and thymus tissues were taken, genome DNA of thymic lymphomas cell was extracted by Promega DNA purification kit, PCR-restriction fragment length polymorphism (RFLP) was used to examine the single nucleotide polymorphism（SNP）of rs51048361（C/T）of c-myc gene,rs30221756（A/G）and rs30161609（T/C）of k-ras gene. Results The thymic lymphomas incidence induced by ⁶⁰Co exposuring in two kinds of mice was different,the genotypes of rs51048361 site were C/C and T/T,the genotypes of rs30221756 site were A/A and G/G,the genotypes of rs30161609 site were T/T and C/C.Conclusion After ⁶⁰Co exposuring,the genotypes of rs51048361,rs30221756 and rs30161609 in C57BL/6J and BALB/c with different radiosusceptibility are different,so it is inferred that there is relevance between the polymorphism of c-myc and k-ras gene and thymic lymphomas induced by ionizing radiation.

Abstract:Objective To study the effects of low dose radiation(LDR) on cell cycle and the expression of its related proteins of HCT-8 cells and provide theoretical basis for clinical application of LDR.Methods Human colon carcinoma cells(HCT-8) cultivated in vitro were divided into seven groups:sham radiation group(0 mGy),LDR groups(25,50,75,100 and 200 mGy) and high dose radiation group(1000 mGy).The proliferation rate was detected with the method of cell count and MTT, the ratios of G₀/G₁,S,G₂/M in cell cycle were determined with flow cytometry after LDR,The cell cycle and expressions of related signal proteins were analyzed with protein assay system.Results The results of cell count and MTT showed that there were no significant differentes of proliferation rate of HCT-8 cells between 25,50,75,100,200 mGy LDR groups and sham radiation group(P>0.05);compared with high dose radiation group,there were significant differences(P<0.05).The result of flow cytometry showed that the ratio of G₀/G₁ phase of HCT-8 cells increased (P>0.05),the ratio of S phase decreased significantly(P<0.05),and the ratio of G₂/M phase increased obviously(P<0.05) 12 and 24 h after 75 mGy radiation compared with sham radiation group.There were no significant differences of the ratios of G₀/G₁,S,and G₂/M phases of HCT-8 cells 48 h after radiation compared with sham radiation group(P>0.05).The protein assay result indicated that the expressions of AKt,PCNA,p27,CDK2,cyclin E,EGFR,ERK1/2,p-ERK,p-GSK-32/β in HCT-8 cells after LDR decreased compared with sham radiation group.Conclusion LDR has no stimulating effect on HCT-8 cells.However,to some extent LDR suppress the expressions of some proteins related to proliferation and cell cycle.

Abstract:Objective To construct the vector for β-galactosidase (β-gal) gene expression regulation with tetracycline-regulated gene expression system,and to control β-galactosidase gene expression in hepG2 cells with the vector.So that offer a experimental base for regulating gene expression for liver cancer gene therpy with this system.Methods The primers for explanding two tetracycline operator (TetO2) gene according to the gene nucleotide sequences of TetO2 gene and pcDNA3.1 vector were designed.TetO2 gene was amplified by PCR with pcDNA3.1 plasmid as template.The TetO2 PCR products were cloned into pcDNA3.1 and the vector was named as pcDNA3.1-TetO2.The pcDNA3.1-Teto2 with TetO2 PCR products sequence was analyzed by ABI3130 sequencing analysis.Then β-gal gene was cloned into pcDNA3.1-TetO2,the vector was named as pcDNA3.1-TetO2-β-gal.The pcDNA6/TR plasmid vector with tetracycline repressor(TR) was transfected into HepG2 cells by Lipotap,and the stable transfection cells were screened by blasticidin.TR gene expression was detected by RT-PCR in HepG-2 cells with and without pcDNA6/TR plasmid transfection.The pcDNA3.1-TetO2-β-gal plasmid vector was transfected into HepG2 cells with and without pcDNA6/TR plasmid transfection,and 3 to 4 d later, these transfected gene cells were treated with doxycline（4 mg•L^-1）.After 48 h,β-gal gene expression was detected with β-gal cell staining.Results The pcDNA3.1-TetO2 sequencing analysis results showed that a cassette was made for a cytomegalovirus-type 2 tetracycline operator (TetO2)-TetO2 promoter in pcDNA3.1-Teto2.The double digestion reslut of pcDNA3.1-TetO2-β-gal plasmid vector demonstrated that β-gal gene was successfully cloned into pcDNA3.1-TetO2 vector .The RT-PCR result of TR gene showed TR gene could be expressed in HepG2 cells with pcDNA6/TR plasmid transfection and TR gene didn’t express in HepG2 cells without pcDNA6/TR plasmid transfection.β-gal gene expressed in HepG2 cells without pcDNA6/TR plasmid transfection,but didn’t express in HepG2 cells with pcDNA6/TR plasmid transfection.However,β-gal gene expression could be induced with doxycline in HepG2 cells with pcDNA6/TR plasmid transfection. Conclusion The tetracycline-regulated gene expression system vector for regulating β-gal gene expression is successfully constructed and the vector system can control β-gal gene expression in HepG2 cells.

Abstract:Objective To discuss a new method for linking two subunit genes p35 and p40 of IL-12 by a linkage peptide,so that offer a experimental base for studying the biological function and application of IL-12. Methods Two　 bovine elastin motifs (ten amino acid) genes were used as linkage peptide for linking two subunits gene. The primers for explanding p35 and p40 genes of IL-12 were designed according to the gene nucleotide sequences of p35 and p40 genes and linkage peptide.The part linkage peptide gene sequences and Kpn Ⅰ digestion site were arranged separately to antisense primer of p40 gene and sense primer of p35 gene.The p35 and p40 genes were explanded by PCR.The PCR products of p35 and p40 genes were digested with Kpn Ⅰ restriction endonuclease.The two digestion products were ligated by T4DNA ligase after two digestion products were purified.The ligated products were explanded by PCR with sense primer of p40 gene and antisense primer of p35 gene.The PCR products were identified by restriction endonuclease digestion with Kpn Ⅰ.The PCR products were cloned into T clone vector,and the positive clones were picked out,then the T clone vector was identified by restriction endonuclease digestion.The nucleotide sequences of ligated products were judged by sequencing analysis. Results The p40 gene product that nucleotide number was 1 000 bp and the p35 gene product that nucleotide number was 600 bp were obtained by PCR.The ligated products of two subunits of IL-12 that nucleotide number was 1 600 bp were obtained.Two gene segments were obtained by digesting the ligated products with Kpn Ⅰ endonuclease.The sequencing analysis results showed that the sequence of ligated products was same as the gene nucleotide sequences of p35 and p40 genes and linkage peptide reported in GenBank.Conclusion The two subunits of IL-12 are successfully linked with linkage peptide by using this method.A new,simple,convenient and effective method for linking two subunits of IL-12 is found,and the paper offers a experimental base for farther study on the biological function and application of IL-12.

Abstract:Objective To observe the effect of Ginsenoside on mouse auricle microcirculation disorder induced by adrenaline．Methods Forty mice were equally divided into 4 groups(n=10)：control group，Ginsenoside Rb₁ treated group，Ginsenoside Re treated group and Ginsenoside Rg₁ treated group．Ginsenoside Rb₁，Re or Rg₁ were separately given intraperitoneally in the mice per day at dose of 10 mg•kg^-1 for 4 d．The microcirculation disturbance in mice was brought by rapid injection of adrenaline．The micro vascular diameter，the blood flow speed and micrangium crossing net patefaction number in auricle of mice at normal condition and in the 10,20,30 min after injection of adrenaline were observed by microcirculation microscope with analytical system．Results Compared　 with control group,Ginsenoside Re or Rg₁ could expend the narrower diameter of the microangium (P<0.05 or P<0.01)，quicken the slower blood flow speed induced by adrenaline (P<0.05 or P<0.01)，and increase the micrangium crossing net patefaction number (P<0.05)．Ginsenoside Rb₁ possessed no significant effect on microcirculation disturbance by adrenaline．Conclusion Ginsenoside Re or Rg₁ can relieve the contract of blood vessels obviously induced by adrenaline．

Abstract:Objective To construct the eukaryotic expression plasmid of recombinant human hypoxia-inducible factor-1α(HIF-1α) and get ready for the coloning and expressing of HIF-1α gene. Methods The total RNA was isolated from blood cells,and cDNA library was constructed by reverse transcription PCR method.PIREGFP containing EGFP fragment and cDNA were used as templet,the three designed primers (EGFP-linker,HIF-1α upstream and HIF-α downstream)were put into,after amplification the target gene fragment was inserted into the shuttle vector T ,and transfected with E.coli　 DH5α,the bacterial colonies containing recombinant plasmids were identified by LB/KANA-agar plate,and the recombinant plasmids were extracted and purified.All sequences amplified by PCR were confirmed by complete sequencing.The correct sequences were cloned into the pVAX1 vector. Results The amplified fragments were about 870,1 199,672 bp by PCR,they were inserted into the shuttle vector T and sequenced. The result of sequencing was identical with that provided by GenBank.The final plasmid about 1 800 bp was obtained by the identification of enzyme digestion and it had same molecular size as expected. Conclusion The eukaryotic expression plasmid pVAX1-EGFP-linker-HIF-1α of recombinant human HIF-1α is successfully constructed.

Abstract:Objective To investigate the effect of IL-12 on mediator release from mast cells and the potential signal transduction pathways correspondingly.Methods P815 cells were challenged with various concentrations of IL-12.The supernatants were collected and analyzed by enzyme-linked immunosorbent assay(ELISA) to detect the quantity of released IL-13,IL-6 and histamine.The cells were treated and analyzed by cellular activation of signal ELISA(CASE) to detect phosphorylation of ERK and P38.Results After incubated with different doses of IL-12(0,1.0，10.0 and 100.0 μg•L^-1),the IL-13 release of P815 cells increased with the concentration of IL-12 compared with medium alone control (P<0.05);but there were no significant differences of IL-6 release and histamine release compared with mediun alone control.After P815 cells were incubated with PD98059 and U0126 prior to IL-12,the percentage of intracellular phosphorylation of ERK and IL-13 release decreased significantly compared with groups without inhibitor (P<0.05) .After P815 cells were incubated with SB203580 prior to IL-12,the percentage of intracellular phosphorylation of P38 and IL-13 release didn’thange compared with groups without inhibitor.Conclusion IL-12 induced IL-13 release from P815 cells is likely through activation of ERK signalling pathway.

Abstract:Objective To investigate the methods of isolation，culture，identification and labeling of mesenchymal stem cells(MSCs) in vitro　and lay a foundation for further study on intervention of MSCs on immunologic rejection of organ transplantation. Methods MSCs were isolated and cultivated by adherent methods . The expressions of CD90 and CD45 of cells were analyzed by using flow cytometry in order to identify MSCs．The third generation of MSCs were labeled by DAPI,the labeling efficiency was detected．Results Primary cultured MSCs adhered to plastic surface within 48 h and reached 90％ confluence within 7－10 d ．Flow cytometry showed that the positive rates of CD90 and CD45 of MSCs at third generation were 99.8% and 6.8%. MSCs expressed CD90 but no CD45．All of the MSCs after labeling by DAPI showed blue fluorescence by immunofluoroscope. DAPI labeling was sensitive and highly efficient to MSCs．Conclusion Adherent method is simple and easy to isolate and cultivate MSCs and it can serve as a routine method．DAPI labeling can be used as a efficient method to label MSCs．

Abstract:Objective To investigate the effects of rhBMP₂ on the differentiation of human dental pulp stem cells and expression of Delta protein in vitro. provide a theoretical basis for research on human dental pulp stem cells. Methods Monocell suspension was separated from the human adult dental pulp with collagenase Ⅰ and dispase digestion.Clonogenic cells were observed under light microscope and the expressions of surface markers were determined with immunofluorescence. divided into experimental group (containing 50 μg•L^-1rhBMP₂ in the culture medium) and negative control group (containing culture medium only).alkaline phosphatase and the expression of Delta proterin of the cells at at passage 5 were detected.Results The human dental pulp stem cells showed colony growth,the nestin and vimentin staining were positive by immunohistochemical staining.STRO-1 was positive in cells by immunofluorescence.Compared with negative control group, the activities of alkaline phosphatase increased after treatment with rhBMP₂ for 7,14 and 21d(P<0.01),the expression of Delta protein was higher after treatment with rhBMP₂ for 21d by westem blotting (P<0.05). Conclusion The cultured human dental pulp stem cells have have the characteristics of stem cells.rhBMP₂ may increase the activity of alkaline phosphatase and up-regulate the expression of. Delta protein may be involved in the differentiation process of Human dental pulp stem cells in to odontoblasts like cells.

Abstract:Objective To investigate the expressions of hypoxia inducible factor-1α（HIF-1α） and vascular endothelial growth factor （VEGF） in vessels and muscle of ischemia lower limbs in patients with arteriosclerosis obliterans(ASO) in order to provide a theoretical basis for HIF-1α gene therapy in ischemic disease.Methods The ischemia lower limbs from 15 patients sufferring from ASO were used in the study with those from 3 normal individuals as control.The expressions of HIF-1α and VEGF in vessels and muscle were detected by immunohistochemistry (SP).The neovessels were marked with CD34,and microvessel density(MVD) was demonstrated by counting the number of positive cells of blood vessel endothelium marked CD34.Results The expressions of HIF-1α and VEGF in vessels and muscle of ischemic limbs were augmented obviously,accompanying with increase of MVD,especially in the posterior tibial artery(PTA).Density of HIF-1α,VEGF and MVD in the PTA were 932.5±545.2,354.5±75.8,and 38.4±8.4 separately.On the other hand,the expressions of HIF-1α and VEGF as well as MVD were less obvious in superficial femoral artery,anterior tibial artery,the vessels of ischemic muscular tissue of calf,the muscular tissue around necrotic tissue of feet compared with those in PTA.In ischemia vessels,the expressions of HIF-1α and VEGF and MVD increased obviously with the ischemia degree.On the contrary,the expressions of HIF-1α and VEGF and MVD in ischemia muscle were not related to ischemia degree.The expressions of HIF-1α and VEGF in ischemia muscle were less than those in ischemia vessels.Conclusion The　 expressions of HIF-1α and VEGF both in vessels and in muscle tissues of ischemia limbs in ASO patients are higher than those in normal tissues.The expressions of HIF-1α and VEGF in ischemia muscle are obviously lower than those in ischemia vessels.

Objective To investigate the relationship between the protein expression and the methylation in promoter region of death-associated protein kinase DAPK gene and oral squamous cell carcinoma (OSCC) and study the mechanism of the DAPK protein expression changes and the gene therapy method in OSCC.Methods The methylation rates in promoter region of DAPK gene in 23 cases of normal oral mucous membrane and 53 cases of OSCC tissues were detected by methylation specific PCR,and the expression rates of DAPK protein were analyzed by immunohistochemistry.Results There was no methylation in promoter region of DAPK gene in normal oral mucous membrane.The exhaustive methylation in promoter region of DAPK gene in 30 cases（56.60%）and partial methylation in 8 cases （15.09%）in OSCC were detected.The total methylation rate was 71.69% in OSCC,there was significant difference compared with normal oral mucous membrane（P<0.01）.The expression of DAPK protein was positive in 23 cases (100%) of normal oral mucous membrane and was down regulated in 36 cases (67.92%) of OSCC tissues,there was significant difference（P<0.01）.There was a significant difference in the methylation rate between down-regulated expression group in OSCC tissues（32/36,88.89%） and normal expression group（6/17,35.29%）.（P<0.01）.Conclusion The DNA methylation in promoter region might be one of the mechanisms that contribute to down-regulation of the expression of DAPK protein.The loss of DAPK gene’s function caused by the methylation in promoter region may relate to the tumorigenesis of OSCC.

Objective To investigate the genetic association between the polymophism of progesterone receptor(PR) gene and susceptibility of uterine leiomyomas.Methods The method of PCR-RFLP was conducted to examine the genotypes of rs1145460 site of PR gene in 165 patients with uterine leiomyomas and 157 healthy women of Han descent. Hardy-Weinberg equilibrium for genotypic distribution was tested using the Chi-square (χ²) goodness-of-fit test.SPSS12.0 was applied to analyze the association between allelic gene and genotypic distribution and uterine leiomyomas.Results The genotypic frequency distribution of rs1145460 was not deviated from Hardy-Weinberg equilibrium in both patient group and control group (P>0.05).The genotypic（C/C,C/T and T/T）and allelic（C and T）frequencies of rs1145460 site had no significant difference between patient and control groups (P>0.05). Conclusion The polymorphism of rs1145460 site of PR gene may be not associated with the pathogenesis of uterine leiomyoma in Han women from north china.

Objective To investigate the expressions of CD40 and ICAM-1 in primary hepatocarcinoma and clinical significance and analyze their relationships with carcinogenesis and progression of liver cancer. Methods S-P immunohistochemistry was used to detect the expressions of CD40 and ICAM-1 in 40 cases of hepatocarcinoma and 25 cases of normal liver tissue (distance from cancer tissues≥2 cm). Results The positive expression rates of CD40 and ICAM-1 were 47.5% and 82.5% in liver cancer tissue,which was significantly higher than those in normal liver tissue (P<0.01).The positive expression rate of CD40 in liver cancer tissue was associated with lymph node metastasis(P<0.05),but not associated with histological grade(P>0.05).The positive expression rate of ICAM-1 was associated with lymph node metastasis as well as histological grade(P<0.01 or P<0.05). Conclusion There are abnormal expressions of CD40 and ICAM-1 in liver cancer.It may be a reliable indicator to judge the invasion and metastasis and prognosis of primary hepatocarcinoma and provide experimental evidence for biological and clinical research.

Objective To investigate the association between angiotensin Ⅰ converting enzyme(ACE) gene 　polymorphism and type 2 diabetic nephropathy.Methods In 80 patients with diabetic nephropathy(DN),60 patients with type 2 diabetes mellitus (DM)and 60 health persons (control),the white cell template DNA of peripheral blood were extracted,the ACE gene (the 16th intron of 287 bp I/D) polymorphism was determined by polymerase chain reaction (PCR),then 1.5% agarose gel electrophoresis was performed,the results were observed under ultraviolet light,the genotypes of each case were confirmed,the genotypic frequency and allelic frequency of ACE gene were compared between three groups with Hardy-Weinberg inherit quilibrium laws and Chi-square criterion.Results The distribution of genotypic frequencies of ACE was not deviated from the Hardy-Weinberg equilibrium;there were no remarkable differences of the frequency distributions of I I,ID and DD of ACE between three groups (χ² =1.10，P>0.05),and the frequency distributions of I and D had no markedly differences between three groups(χ² =0.84，P>0.05).Conclusion There is no significant correlation between ACE gene polymorphism and type 2 diabetic nephropathy.

Objective To explore the cellular location of ER subtypes (ERα and ERβ) in normal human mammary gland and provid the foundation for exploring the relationship between ERα,ERβ and development of normal mammary gland. Methods Using immunohistochemistry,the expressions and cellular distribution of ERα and ERβ in the normal tissues of 16 patients who had accepted reduction mammoplasty specimens in the Affiliated Hospital of University Laval were detected.Results ERα immunoreactivity was detected in the nuclei of epithelial cells lining lobules and ducts.The positive cells located in the inner layer of the two epithelial layers in the lobules and intralobular ducts,but in the interlobular ducts the positive cells located in the outer layer.Although ERβ was also seen in these cells,there was irregularity of its distribution.Weak to moderate cytoplastic staining of ERβ in epithelial cells of lobules and ducts were fonud.Occasional nuclear staining was seen in the stromal cells,endothelial cells and lymphocytes.The percentage of ERβ positive expression in the epithelial cells was much higher than that of ERα,there was markedly difference between them (t=29.789，P<0.01 ).The positive expression rate of ERβ in the mammary gland was much higher than that of ERα,there was markedly difference between them (χ²=8.127,P＜0.05). Conclusion ER subtypes have distinct distribution patterns in the normal mammary gland.The widespread distribution of ERβ suggests that it may be the dominant ER in the mammary gland.

Objective To explore potential biomarker candidate(s) in stage Ⅱ breast infiltrating ductal carcinoma with negative estrogen receptor (ER^-) and to discuss the significance of the identified proteins in tumor invasion and metastasis.Methods Two-dimensional polyacrylamide gel electrophoresis (2-DE) in combination with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-MS) and bioinformatics analysis were performed to investigate differential proteins of the infiltrating ductal carcinoma at stage Ⅱand adjacent normal breast tissues from 5 breast cancer patients with negative estrogen receptor.Results The number of the tested protein spots from 5 adjacent normal breast tissues was from 779 to 1 328.The number of the tested protein spots from 5 breast cancer tissues was from 988 to 1 453.The correspondent spots was from 310 to 774. Three differential proteins (S1,S2,S3) from the 2-DE gel images of 5 patients’cancer tissues and adjacent normal tissues were obtained.Three protein candidates were identified: immunoglobulin lambda chain variable region (S1),collagen alpha-3 (Ⅵ) chain precursor (S2) and S100 calcium binding protein A11 (S3).Proteins S2 and S3 were found to be up-regulated in all 5 cancer tissues (abundance changed more than 2 folds).Conclusion Proteins can be obtained efficiently from tissues under the condition of optimized protocols of tissue 2D-PAGE;Collagen alpha-3 (Ⅵ) and S100 calcium binding protein are likely involved in invasion and metastasis in the stage Ⅱ breast infiltrating ductal carcinoma with negative estrogen receptor.

Objective To explore the expressions of IgA,IgM,IgD,IgE in ovarian epithelial carcinoma and their correlations with pathological grades of ovarian epithelial carcinoma.Methods The expressions of IgA,IgM,IgD,IgE in ovarian epithelial carcinoma tissue microarrays (including 177 cases of ovarian epithelial carcinoma)and 50 cases　 of ovarian benign tumor tissues were detected by immunohistochemistry.Results The expressions of IgA,IgM,IgD,IgE in ovarian epithelial carcinoma tissues were significant higher than those in ovarian benign tumor epithelial tissues (P<0.001). In ovarian epithelial carcinoma there were significant positive correlation etween pathological grades and the expressions of IgA,IgM,IgD and IgE(r =0.422，P<0.001;r =0.460，P<0.001);r =0.458，P<0.001;r =0.593，P<0.001), the expressions of IgA,IgM,IgD and IgE were more stronger with raising of pathological grades.Conclusion IgA,IgM,IgD,IgE express highly in ovarian epithelial carcinoma;the abnormal expressions of IgA,IgM,IgD,IgE in ovarian epithelial carcinoma indicate that they are related to the occurrence and development of ovarian epithelial carcinoma.

Objective To detect the expressions of hypoxia inducible factor - 1α (HIF-1α) and microvesseldensity (MVD)in ovarian epithelial tumor tissues ,and investigate the effect of HIF-1α and MVD in development of ovarian epithelial tumor.Methods 237 patients with ovarian epithelial tumor were selected,including 79 cases of serous cystadenoma, 12 cases of borderline serous cystadenoma,51 cases of serous cystadenocarcinoma, 67 cases of mucinous cystadenoma,9 cases of borderline mucinous cystadenoma and 19 cases of mucous cystoadenocarcinoma.Serous cystadenocarcinoma and mucous cystoadenocarcinoma were divided into different stages according to FIGO operation-pathology,stage Ⅰ was 13 cases,stage Ⅱ 26 cases,stage Ⅲ 19 cases and stage Ⅵ 12 cases.The expressions of of HIF-1α and MVD in ovarian epithelial tumor tissues were detected with immunohistochemistry.The expressions of HIF-1α and MVD were compared in benign tumor,borderline tumor and malignant tumor,as well as in different stages of malignant tumor.The relationship between the expression of HIF-1α and MVD value was also analyzed.Results The positive expression rate of HIF-1α in borderline serous cystadenoma group was significantly increased,compared with ovarian benign tumors (serous cystadenoma and mucinous cystadenoma groups)(P<0.05).The positive expression rates of HIF-1α in ovarian serous cystadenocarcinoma and mucous cystoadenocarcinoma were significantly superior to the benign tumors(P<0.05).The expressions of MVD in borderline serous cystadenoma and borderline mucinous cystadenoma groups were significantly higher than that in ovarian benign tumors(P<0.05).The expressions of MVD in serous cystadenocarcinoma and mucous cystoadenocarcinoma groups were sinigicantly higher than those in benign tumor group and borderline tumor group(P<0.05).The positive expression rates of HIF-1α and MVD were raised up with the process of the stage of FIGO operation-pathology.And there was positive correlation between HIF- 1α positive expression and MVD (r=0.540，P<0.01).Conclusion The overexpression of HIF-1α in ovarian epithelial tumor is closely related with angiogenesis,which may play an important role in the occurrence and development of ovarian epithelial tumor.

Abstract:Objective To study the expression of CD4⁺CD25^high Foxp3⁺ regulatory T cells (Tregs) in peripheral blood monouclear cell(PBMC) of patients with cancer,and explore the significance of Tregs in pathogenesis of cancer.Methods The proportions of CD4⁺CD25⁺/ CD4⁺,CD4⁺CD25^high / CD4⁺T cells in PBMC were examined with flow cytometry in 61 patients with cancer at different stages,and the expression levels of Foxp3⁺ in CD4⁺CD25⁺ and CD4⁺CD25^high T cells were analyzed,and compared with 32 normal control samples.Results Compared　 with normal controls,the proportions of CD4⁺CD25⁺/ CD4⁺,CD4⁺CD25^high / CD4⁺T cells in PBMC of patients with cancer at stageⅠand stage Ⅱ had no significant difference(P>0.05);but in those patients at stage Ⅲ and stage Ⅳ,they were higher than those in normal controls,the difference was significant(P<0.05).The proportions of CD4⁺CD25⁺/ CD4⁺,CD4⁺CD25^high / CD4⁺T cells were higher in PBMC of patients with cancer with higher histophathological grade.The expression levels of Foxp3⁺ in CD4⁺CD25⁺ and CD4⁺CD25^high T cells in patients with cancer were also higher than those in normal controls(P<0.01),the differences were significant(P<0.01). Conclusion The increase of Tregs in patients with cancer may be related to tumor progression and it shows immunological abnormality in patients with cancer.The regular detection of CD4⁺CD25⁺,CD4⁺CD25^high and CD4⁺CD25^high Foxp3⁺ in PBMC will contribute to understanding the patient’s condition and judging prognosis.

Objective To investigate the association between CYP1A1-MspⅠ gene polymorphism and genetic susceptibility of breast cancer.Methods CYP1A1-MspⅠ gene polymorphism in 160 patients with breast cancer and 124 controls were detected by PCR-RFLP.The frequencies of genotype and allele were analyzed.Results The distribution frequencies of genotype of CYP1A1[-MspⅠ site in case group were TT45.0％,TC46.3％,CC8.8％,and the frequencies of allelic gene were T（68.1％） and C（31.9％）. While the distribution frequences of genotype of CYP1A1-MspⅠ site in control group were TT61.3％,TC35.5％,CC3.2％,and the frequences of allelic gene were T79.0％,C21.0％.The ratios of genotypes in each group had no significant difference（P>0.05）；The frequencies of allelic gene of T and C in each group had significant difference（P<0.01）.ConclusionCYP1A1-MspⅠ gene polymorphism may be linked with genetic susceptibility of breast cancer in this area.

Objective To understand the prevalence,distributions and influencing factors of obsessive compulsive neurosis in college students and provide evidence for intervention method and etiology study.Methods Stratified-proportional　 sampling method was conducted in 2 046 college students,who were interviewed using the third version of the World Health Organization Composite International Diagnostic Interview and diagnosed by ICD-10，1 869 students finished the interview and 1 843 qualified papers were collected.Results The lifetime prevalence of obsessive compulsive neurosis in college students was 17.1%,12-month prevalence was 9.9% and 30-day prevalence was 2.5%;The male’s lifetime prevalence was significantly higher than the female’s(χ²=6.107,P=0.013);The lifetime prevalence of obsessive compulsive neurosis had significant differences in different grades(χ²=16.223,P=0.001),and the junior’[KG-*3]s was lower than the senior’s;Through multi-factor analysis,seven influencing factors were found: grade,gender,mood of father,mood of mother,quarrels between parents,ignored care from parents and the relationship with friends in social net. Conclusion The lifetime prevalence of obsessive compulsive neurosis in college students is higher than others,and the influencing factors are grade,gender,mood of father,mood of mother,quarrels between parents,ignored care from parents and the relationship with friends in social net.These results suggest that colleges,families and the society should pay more attention to obsessive compulsive neurosis in college students and take positive measures to prevent it.