Abstract:Objective To explore the effect of N-acetylcysteine (NAC) on splenocyte apoptosis and immune associated factors in rats with diabetes mellitus(DM),to provide the theoretic evidence for prevention of the complication of DM and therapy of DM.Methods The rats were divided into control group,DM group and NAC+DM group.The DM rat model was induced by intraperitoneal injection of streptozotocin.At the fourth weekend after giving NAC through intragastric administration,the apoptosis,cell cycle and TCRαβ% of splenocytes were detected with flow cytometry,Annexin V/PI double-staining,PI single-labeling and TCRαβ antibody-PE respectively;and the IL-2 contents in serum and splenocyte culture supernatant were measured with ELISA.Results As compared with control group,the apoptotic rate,TCRαβ% of splenocytes and the serum IL-2 content in rats in DM group increased significantly (P<0.001),and both the percentage of splenocytes in G₀/G₁ and G₂/M phases increased significantly (P<0.05),and the percentage of splenocytes in S phase and the IL-2 content in splenocyte culture supernatant decreased significantly (P<0.05); the apoptosis of splenocytes did not decrease significantly,and so was that of cell cycle. As compared with DM group,the apoptotic rate,TCRαβ% of splenocytes and the serum IL-2 content in DM+NAC group decreased significantly (P<0.001),and the percentages of splenocytes in both G₀/G₁ and G₂/M phases decreased significantly (P<0.05),and that in S phase and IL-2 content in splenocyte culture supernatant increased significantly (P<0.05).Conclusion NAC could decrease the increase of splenocyte apoptosis rectify the cell cycle delay of splenocytes and regulate the immune factor imbalance induced by DM.

Abstract:Objective To explore the feasibility of orthotopic transplantion tumor model of hepatoma in mice and evaluate the biological features.Methods 5×10⁶ H22 cells were inoculated into the liver of mice after anesthetizing.The liver,spleen,lung and kidney were taken from the mice 13 d after inoculation(n=8) or from the died mice(n=9) for morphological evaluation.Proliferating cell nuclear antigen(PCNA) and factor Ⅷ related antigen(FⅧRag) were analyzed with immunohistochemical staining.Results The average survival time of the mice was 18.8 d.Until the 13th day after inoculation,the successful rate of orthotopic transplantation tumor (model of liver cancer) was 100%(8/8),spontaneous lung metastatic rate was 37.5%(3/8).Fifty percent of mice(4/8) produced ascites,and the average amount of ascites was （6.25±3.67）mL.The average size of the tumor was （0.35±0.14） cm³.In the natural died mice,spontaneous lung metastatic rate was 100%（9/9）,100% of mice (9/9)produced ascites. The average volume of acites was （10.88±1.92）mL,which was evidently more than that of the 13th day（P<0.01）.The average size of the tumor was (0.61±0.31) cm³, which was significantly bigger than that of the 13th day（P<0.05）.When the mice died,most of tumor nodules were single and the cellular 〖JP2〗atypia was apparent.The proliferation index and microvessel density of tumor tissue were 71.39%±〖JP〗20.74% and (39.25±13.71)/400 high magnification respectively.Conclusion The successful rate of orthotopic transplantation tumor model of liver cancer is high.The tumor possess high lung metastatic rate and plenty of microvessels and strong proliferative ability.

Abstract:Objective To investigate the influence of HIWI gene silencing in biological behavior of T24 cells and explore the possibility for HIWI gene to be used as the molecular target of inhibiting bladder carcinoma cell proliferation with gene transfection and RNA interference (RNAi) technique.Methods T24 cells were divided into transfection group with pGenesil-2-HIWI,transfection group with pGenesil-2-HIWI2263,transfection group with pGensil-2-control,and two control groups transfected with PEI only, and PBS only, respectively.T24 cells were transfected with shRNA expression vectors targeting HIWI gene by PEI,and the cell proliferation and cell cycle were measured by MTT assay and FCM.Results At the time of post-transfection 24 h,the inhibitory rate of cell proliferation in transfection groups were 32.60% and 26.09%,they were lower than that in control group (3.54%). At the time of post-transfection 48 h,the percentages of cells at S phase in transfection groups were (29.39± 3.27)% and (30.87±10.88)%,they were lower than that in control group (39.36%±2.09%) (P<0.01),and the percentages of cells at G₂/M phase in transfection groups were (9.39±0.80)% and (11.46±1.53)%,they were higher than that in control group (5.57%±0.40%) (P<0.05).Conclusion HIWI gene silencing has inhibitory effect on the proliferation of T24 cells.HIWI gene could be a molecular target of inhibiting bladder carcinoma cell proliferation,and HIWI gene silencing has a protential treatment value in inhibiting bladder carcinoma cell proliferation.

Abstract:Objective To culture human mesenchymal stem cells (hMSCs) and study the ultrastructure of their myogenic differentiated cells in vitro,and provid the theoretical foundation for hMSCs differentiation into cardiomyocytes (CMs). Methods hMSCs were cultivated and expanded by adhereing peculiarity.The phenotypes of hMSCs were identified by fluorescent - activated cell sorter(FACS).With good growth characteristics and high purity,the fifth passage hMSCs were treated for 24 h with 5-azacytidine (5-Aza,10 μmol•L^-1) to induce cellular differentiation.The expression of desmin,identified by immunohistochemistry,was used to identify cardiac muscle differentiation.The morphology and ultrastructure of treated hMSCs were observed by inverted microscope and transmission electron microscope. Results The cultured cells did not express CD34 and expressed a high level of CD44 by FACS,which indicated that they were hMSCs.The morphology of hMSCs treated with 5-Aza under inverted microscope changed from fusiform to polygon or astrocyte,and the induced hMSCs demonstrated positive staining for desmin.With transmission electron microscope,the ultrastructure of the treated hMSCs was visible.The magnitude of cells was not identical,the surface of some cells had abundance of microvilli.Intracytoplasm had rich organelles,mitochondrium and rough endoplasmic reticulum.In the part of cells,the sedimentary hepatin granules were observed and only the myeloid body could be seen in small part of cells.Myofilaments were found in cytoplasm,and between cells there were cell junctions. Conclusion After treated with 5-Aza,hMSCs cultivated and expanded in vitro could differentiate into cardiomyocyte-like cells with CMs’ ultrastructure characteristics.

Abstract:Objective To observe the effect of lipopolysaccharide (LPS) on malignant glioma C6 cells injuried by hydrogen peroxide (H₂O₂),investigate the mechanism maybe involved.Methods The experiment was divided into four groups: control group,100 μmol•L^-1 LPS group,1 000 μmol•L^-1 H₂O₂ group and LPS combined with H₂O₂ treatment group.After C6 cells were treated by drugs for 6 h,the LDH release rate was detected by ultraviolet spectrophotometry with LDH detecting kit,the apoptotic rate of C6 cells was determined by TUNEL,the Bcl-2,Bax,Caspase-3 mRNA levels were detected by RT-PCR and Bcl-2,the Bax,Caspase-3,Cyt-C,and CLIC4 protein levels were detected by Western blotting.Results Compared with control group,the apoptotic rate and LDH release rate increased in LPS group (P<0.05),the expression levels of Bcl-2,Bax mRNA did not change (P>0.05),the expression levels of Caspase-3 mRNA was higher than that in control group (P<0.05),but the Bcl-2,Bax,CLIC4 and cytosolic Cyt-C protein expression levels did not change (P>0.05),the expression level of Caspase-3 protein increased(P<0.05).Compared with control group,the apoptotic rate and LDH release rate increased in 1 000 μmol•L^-1 H₂O₂ treatment group (P<0.05),the expression of Bcl-2 mRNA decreased compared with control group (P<0.05),the expression levels of Bax and Caspase-3 mRNA increased compared with control group (P<0.05); but the expression level of Bcl-2 protein decreased,the Bax,Caspase-3,CLIC4 and cytosolic Cyt-C protein expression levels increased obviously (P<0.05).When compared with H₂O₂ group and LPS group,the apoptotic rate and LDH release rate increased in LPS combined with H₂O₂ treatment group (P<0.05),the expression level of Bcl-2 mRNA decreased,the expression levels of Bax and Caspase-3 mRNA increased (P<0.05); the expression level of Bcl-2 protein decreased,the Bax,Caspase-3 and cytosolic Cyt-C protein expression levels increased obviously compared with control group (P<0.05). and the CLIC4 protein expression did not change(P>0.05).Conclusion LPS can activate Caspase-3 signal pathway,induce the early apoptotic event of C6 cells.Meanwhile,the LPS combined with H₂O₂ can aggravate the injury of C6 cells induced by H₂O₂ through the overactivation of apoptosis relative Bcl-2/Bax and Caspase-3 pathway.The function of mitochondrial chloride intracellular channel CLIC4 seems uneffective.

Abstract:Objective To observe the dose- and time-effect of ionizing radiation in combination with 5-flurouracil (5-FU)　 on the cell cycle uncoupling of EL-4 cell line.Methods EL-4 cells were collected after irradiation with 0,1.0,2.0 and 4.0 Gy X-irradiation and treatment with 5-FU (0.001,0.010,0.100 and 1.000 mg•L^-1) for 0,4,8,16,24 and 48 h. The regularity in the polyloid cells was analyzed by flow cytometry (FCM) following staining cells with propidium iodide (PI). Results As compared with sham-irradiation group,the percentage of diploid EL-4 cells increased significantly at 8-24 h and returned to normal level at 48 h after irradiation with 2.0 Gy X-rays (P< 0.05 or P< 0.01); the percentage of tetraploid cells began to decrease at 8 h and still to remain lower level at 48 h (P< 0.05 or P< 0.01);the percentage of octoploid cells 〖JP3〗decreased obviously at 16-24 h (P< 0.05). As compared with sham-irradiation group,the percentage of diploid EL-4 cells increased significantly 16 h after irradiation with 1.0-4.0 Gy X-rays (P< 0.05 or P< 0.01),while that of tetraploid cells decreased in a dose-dependent manner(P< 0.01),the percentage of octoploid cells had no marked changes.As compared with 0 mg•L^-1 group,the percentage of diploid cells decreased obviously at 16-48 h after treatment with 0.10０ mg•L^-15-FU(P< 0.01);the percentage of tetraploid cells increased significantly at 8-24 h and returned to normal level at 48 h;the percentage of octoploid cells increased obviously at 4-16 h (P< 0.05).As compared with 0 mg•L^-1 group,the percentage of diploid cells decreased significantly 16 h after treatment with different doses 5-FU (P< 0.05 or P< 0.01); while that of tetraploid cells increased significantly (P< 0.05 or P< 0.01),they displayed in a dose-dependent manner in 0.001-1.000 mg•L^-1; the percentage of octoploid cells increased significantly after treatment with 0.010 and 0.100 mg•L^-1 5-Fu (P< 0.01).Conclusion The cell cycle uncoupling of EL-4 cell line couldn’ t be induced by 1.0-4.0 Gy X-rays,but could be induced by 0.010 and 0.100 mg•L^-1 5-FU.

Abstract:Objective To study the inhibitory effect of hammerhead ribozyme targeting connective tissue growth factor (CTGF) on collagen I synthesis and cell cycle progression of human hepatic stellate cell line (LX-2) cells.Methods Hammerhead ribozyme cDNA targeting CTGF mRNA plus two self-cleaving sequences were inserted into pTriEx2 vector to construct a recombinant vector pTriCTGF-Rz.LX-2 cells were transfected with either pTriEx2 or pTriCTGF-Rz and further stimulated with or without TGF-1. There were five groups in the experiment:control group,pTriEx2 group,pTriCTGF-Rz group,pTriEx2 plus TGF-β1 group,and pTrCTGF-Rz plus TGF-β1 group.Semi-quantitative RT-PCR was used to detect the levels of CTGF mRNA and collagen Ⅰ mRNA.ELISA and flow cytometry were used to detect the levels of collagen Ⅰ secretion and cell cycle.Results Transfection of pTriCTGF-Rz into LX-2 cells reduced the CTGF mRNA and collagen Ⅰ mRNA levels as well as collagen 　Ⅰ protein level compared with pTriEx2 group(P<0.05).TGF- 1-induced CTGF mRNA and collagen Ⅰ mRNA transcription were decreased in LX-2 cells transfected with pTriCTGF-Rz compared with control groups (P<0.05).Furthermore,in the LX-2 cells transfected with pTriCTGF-Rz,the proportion of cells at G₀-G₁ phase increased (P<0.05)　 and that of cells at S phase decreased(P<0.01) compared with control group.Conclusion The hammerhead ribozyme targeting CTGF has inhibitory effects on human hepatic stellate cell-mediated fibrosis and it may become another candidate for gene therapy in the future.

Abstract:Objective To investigate the changes of expressions of angiogenin-1 (Ang-1) and -2 mRNA in the ischemic brain at different time points,find their regularities and discuss their mechanisms of action after cerebral ischemic stroke.Methods All the rats were divided into 3 groups: control group (group C),sham-operation group (group S),and ischemia group (group I).Rats in group I were divided into 7 subsets,they were 1,3,6,12,24,48 and 72 h after middle cerebral artery occlusion(MCAO) (n=7 per time point).The mRNA levels of Ang-1 and Ang-2 were measured by reverse transcription polymerase chain reaction (RT-PCR).Results In the brain tissues of the rats in group C and group S,the expression levels of Ang-1 mRNA were moderate, the expression levels of Ang-2 mRNA were extremely low,there were no significant differences between two groups(P>0.05). In group I,the mRNA levels of Ang-1 were lower than those in group C during 3-6 h after onset of ischemia(P<0.01),and were higher than those in group C during 48-72 h after MCAO(P<0.01).After MCAO,the expression level of Ang-2 mRNA was higher than that in group C at 3 h after onset of ischemia,the level peak at 12 h,and last for up to 72 h after ischemia(P<0.01).Conclusion Ang-1,Ang-2 and other specific vessel growth factors may regulate the process of vessel formation together after focal cerebral ischemia,they can enhance blood flow,restrain neuron apoptosis in ischemic penumbra,and promote nerve function to recover.

Abstract:Objective To study the efficient expression of the human recombinant β-amyloid protein 1-42(hAβ_1-42),and produce hAβ_1-42 by optimized fermentation parameters on large scale using Pichia pastoris as host system.Methods On the base of construction of the recombinant expression vector pPICZα-Aβ_1-42,and the obtained recombinant vector was transformed into the Pichia pastoris.hAβ_1-42 was expressed in 80 L fermentor with optimized parameters and the broth was harvested and purified with SP Sepharose and Source TM 30 RPC.Results hAβ_1-42 in the broth could reach maximal yield of 150 mg•L^-1 after induced by methanol for 48 h.The expressed product was purified from the fermentating culture.Analytic results revealed that the relative molecular mass of obtained product was about 4 200.Conclusion hAβ_1-42 can be expressed in Pichia pastoris on large scale.A stable fermentation and purification technology is successfully set up.

Abstract:Objective To study the pathological changes of BALB/c mice infected experimentally with coxsackievirus B3 (CVB/SGM-05) from Sichuan golden monkey.Methods Thirsty male BALB/c mice (4-6 weeks age)were infected with CVB/SGM-05 intraperitoneally(10⁵TCID₅₀/0.1 mL,0.2 mL each mouse),the clinical symptoms were observed.The pathological changes in heart,liver,spleen,lung,kidney,pancreas,stomach,intestines,brain and fat tissues were fixed in 10% formalin and embedded in paraffin,the sections were deparaffinized and stained with hematoxylin and eosin,and then were examined at different time after infection.Results The different degrees of pathological changes could be definitely observed in various organs of mice with myocarditis and conjunctivitis,and the mice died from the 60th hour after inoculation with CVB/SGM-05,the mortality was 80%.The main lesion was fat necrosis in the abdominal cavity of mice.Liver and spleen were swell,and some small grayish-white foci were observed in the liver.The hepatocyte necrosis was severe.There was light congestion in the brain,the mucous membrane was necrosis and abscission on the stomach and intestine.Conclusion Compared with the CVB3-Nancy,many tissues and organs are damaged in BALB/c mice infected with CVB/SGM-05,it suggests that CVB3 from the Sichuan golden monkey is different from the other CVB3 in pathogenicity.

Abstract:Objective To investigate the effect of chloride channel ClC-2 antisense oligonucleotide (AON) on the cell invasion suppression of human glioma U251 cells induced by cisplatin (DDP).Methods The experiment was divided into four groups: control group ( nonsense oligonucleotide),AON group (ClC-2 antisense oligonucleotide),DDP group (cisplatin＋nonsense oligonucleotide),DDP＋AON group ( cisplatin＋ClC-2 antisense oligonucleotide).The mRNA expressions of ClC-2,cyclin D1,MMP-2 and MMP-9 of U251 cells were detected by RT-PCR.The invasion of U251 was measured by Transwell assay.Results Compared with control group,the mRNA expression rates of ClC-2,cyclin D1,MMP-2,MMP-9 in AON group,DDP group,DDP＋AON group were decreased （P＜0.05）.The numbers of invasion cells in control group,AON gr oup,DDP group and AON +DDP group were 81.00±4.58,42.01±4.36,48.33±2.52 and 12.67±5.13,respectively.Compared with control group,the numbers of invasion cells in AON group,DDP group and AON +DDP group were significantly decreased（P＜0.05）.Compared with control group,the rates of cell migration（%） in AON group,DDP group and AON +DDP group were 72.40±2.20,51.48±1.29 and 37.87±1.50,respectively.Cell migration of U251 cells was remarkably decreased after infected with ClC-2 antisense and treated with cisplatin（P＜0.05).Conclusion ①ClC-2 antisense oligonucleotide can inhibit the expression of ClC-2 mRNA in U251 cells. Inhibition of ClC-2 mRNA can suppress the mobility and invasion of U251 cells.③ClC-2 antisense oligonucleotide can facilitate invasion suppression of U251 cells induced by DDP.

Abstract:Objective To investigate the effect of INF-γ on the expression of high mobility group box 1 (HMGB1) and MMP-2 protein in mesangial cells(MC) and its possible mechanism in order to provide basis for treatment of renal injury in systemic lupus erythematosus (SLE). Methods Human MC induced by 5 ng•L^-1 INF-γ were collected in 6,12 and 24 h respectively,as well as cells in normal control group in vitro. The expressions of HMGB1mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry.The expressions of proliferation cell nuclear antigen (PCNA) and MMP-2 protein were detected by immunocytochemistry.ELISA was used to determine the levels of HMGB1 and MMP-2 in supernatant.Results Compared with control group,the PCNA protein expression was up-regulated in INF-γ group,the HMGB1 mRNA and protein expressions in MC obviously increased,the HMGB1 content in supernatant increased significantly.Compared with control group,MMP-2 protein in MC and supernatant in INF-γ group increased; there was positive correlation between HMGB1 and MMP-2 protein in supernatant (r=0.915,P<0.01).Conclusion INF-γ can up-regulate the expression of MMP-2 by promoting synthesis of MC and secretion of HMGB1,it might play an important role in renal injury of SLE.

Abstract:Objective To observe the protective effects of panax quinquefolium 20s-protopanaxtriolsaponins (PQTS) on myocardial ischemia-reperfusion injury in rats in order to provide basis for new drug development.Methods Fifty rats were divided randomly into sham group,ischemia-reperfusion model group and PQTS 12.5,25.0 and 50.0 mg•kg^-1 group(n=10).All animals in PQTS groups were administered with PQTS one time everyday with intraperitoneal injection(i.p) for 3 d，the rats in sham and model groups were given saline. The myocardial ischemia-reperfusion model was induced by 30 min left anterior descending coronary occulusion and 120 min reperfusion in openchest anesthetized rats. The changes of aspartate aminotransferase (AST),lactate dehydrogenase(LDH),MB isoenzyme of creatine kinase(CK-MB),superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content in serum,and the prostacycline(PGI₂) and thromboxane A₂ (TXA₂) levels in plasma were determined.Results Compared with model group,in rats treated with PQTS (12.5,25.0 and 50.0 mg•kg^-1 i.p for 3 d),the MIS was significantly reduced(P<0.01),the AST，LDH and CK-MB activities in serum,the MDA content,and the TXA₂ level in plasma were declined(P<0.05 or P<0.01),while the PGI₂ level in plasma and PGI₂/TXA₂ ratio were increased signficatly(P<0.05).In addition,the SOD and GSH-Px activities in serum were increased markedly(P<0.05 or P<0.01).Conclusion PQTS　 has protective effects on myocardial ischemia-reperfusion injury through enhanceing activity of antioxidase,reducing oxidative damage of free radicals on myocardium and correcting disequilibrium of vasoactive substance.

Abstract:Objective To prepare active constituent from Qianglitianmaduzhong capsule (QLTM) and observe the analgesic effect of active constituent on mice and regulatory effect on macrophages.Methods Mice were randomly divided into five groups: negative control group (normal saline group ),QLTM capsule group (positive control group),active constituent groups with high,middle,and low doses.The mice ware administered by oral gavage every day for 14 d.In hot-plate test,the beginning time and times of licking hind paws within 1 min of mice were observed. In acetic acid-induced writhing test,the beginning time and writhing times within 15 min of mice were observed. The phagocytic ability of peritoneal macrophage was evaluated with chicken red blood cell phagocytosis assay.Results In hot -plate test,the active constituent with high and middle doses prolonged the beginning time of licking hind paws and reduced times of licking hind paws within 1 min (P<0.05 or P<0.01),compared with negative control group.In acetic acid-induced writhing test,the active constituent with high and middle doses prolonged the beginning time of acetic acid-reduced writhing and reduced writhing times within 15 min (P<0.05).Both active constituent and QLTM inhibited the phagocytic ability of mouse peritoneal macrophages,especially in middle-dose group of active constituent,statistically significant difference was observed compared with negative control group (P<0.05).Conclusion The active constituent from QLTM has analgesic effect on mice and can inhibit activation of macrophages.

Abstract:Objective To construct a plasmid expression vector coding for the short hairpin RNA (shRNA) targeting Twist mRNA.Methods Two plasmid expression vectors coding for shRNA targeting 777 and 845 of Twist gene sequence and a control vector containing random DNA fragment were constructed.The recombinant plasmids were identified by PCR,and then transfected separately into bladder cancer cell line T24.The Twist gene silencing effect was detected by RT-PCR and Western blotting.Results The expected band of 400 bp was amplified from the plasmids coding for shRNA by PCR.　By DNA sequencing,it was the same with the insertion element as with the shRNA of synthetic.Transfection of T24 cells expressing Twist gene with the shRNA plasmids resulted in inhibition of Twist mRNA and protein expressions by 90% and 86%,respectively.The shRNA1 had the most obvious effect in Two types of plasmids interference.Conclusion The plasmid expression vectors coding for shRNA targeting Twist mRNA have been constructed successfully,of which pGenesil-shRNA1 most effectively silences Twist gene in T24 cells.

Abstract:Objective To clone human membrane type-1 matrix metalloproteinase (MT1-MMP) gene and construct its eukaryotic expression vector,then detect its expression in human hepatoma cell line HepG2.Methods Full-length human MT1-MMP cDNA was amplified from normal liver by RT-PCR and cloned into pMD18-T simple vector. After sequencing,the fragment was subcloned into the pcDNA3.1 vector and the recombinant eukaryotic expression vector was constructed. MT1-MMP mRNA level of HepG2 cells was evaluated by RT-PCR.Results ① A 704 bp fragment was obtained by PCR,which was the same as the expected fragment.②Two fragments of PCR products (5400 bp and 704 bp) of eukaryotic expression vector pcDNA3.1/MT1-MMP were obtained by EcoRⅠ and BamHⅠ restriction enzyme digestion.The sequencing result of MT1-MMP cDNA was identical with that reported in GenBank.③ MT1-MMP mRNA level of HepG2 cells transfected with the recombinant vector was obviously higher (1.66±0.43) than those of empty vector group (1.21±0.25) and control group (1.19±0.18)(P<0.01).Conclusion The recombinant vector pcDNA3.1/MT1-MMP is successfully constructed and expressed stably in HepG2 cells.

Abstract:Objective To construct the vector of RNA interference(RNAi) targeting survivin gene and observe its effect combined with X-rays radiation on lung adenocarcinoma A549 cell apoptosis. Methods One pair of RNAi sequence targeting survivin gene were designed according to its cDNA sequence reported in GenBank,the recombinant RNAi plasmid pGenesil2-survivin was constructed. After identified by enzyme digestion and sequencing,the pGenesil2-survivin plasmid was trasfeced into A549 cells. In the experiment,normal group,pGenesil2 group,pGenesil2-survivin group,5 Gy irradiation group and pGenesil2-survivin + 5 Gy irradiation group were set up. The apoptosis of A549 cells was measured by flow cytometry with PI/Annexin Ⅴ and TUNEL,the survivin and caspase-3 expressions were measured by Western blotting.Results Two fragments about 389 bp and 4 206 bp were gotten by KpnⅠand EcoRⅠenzyme digestion,they are the same to expected result,the sequencing result was compared to oligonucleotide chain with DNAssist 2.0,they were equal,these indicated the identification of pGenesil2-survivin vector was right; pGenesil2-survivin was transfected into A549 cells for 48 h,the apoptotic percentage in pGenesil2-survivin and 5 Gy X-rays groups increased obviously (P< 0.05),when the both were combined,the effect was more obvious; the Western blotting results appeared that the survivin gray scale/β-actin gray scale in pGenesil2-survivin group was lower than that in normal group (P< 0.01),and the caspase-3 gray scale/β-actin gray scale was higher than that in normal group,and that ratio in pGenesil2-survivin+5 Gy irradiation group was more high (P< 0.01).Conclusion RNAi targeting surviving gene could inhibit survivin protein expression,but enhance caspase-3 protein expression,and promote apoptosis.When it is combined with 5 Gy X-rays irradiation,the promotion of apoptosis is enhanced.

Abstract:Objective To explore the effect of down-regulating survivin expression by RNAi on growth suppression and promotion on apoptosis of human hepatocellular carcinoma cells,in order to supply an effective target and technique for liver cancer gene therapy.Methods The recombinant plasmid of pSU6-si-survivin was constructed,which was transfected into Bel7402 cells.Mock group,negative group and treated group were set up.Using MTT assay the proliferation of transfected cells was investigated,the survivin protein expression level was determined by Western blotting and immunohistochemical staining.Transcription of survivin was detected by semi-quantitative RT-PCR.Flow cytometry(FCM) and AO/EB double dyeing were used to examine apoptosis in transfected cells.Results MTT assay demonstrated that the cell growth inhibition rates(%) of transfected cells in treated group at 24,48 and 72 h were 21.43±1.67，39.07±3.80，and 59.72±7.05，there were significant differences compared with negative group (P<0.01）.The results of RT-PCR and Western blotting showed that the mRNA and protein levels of survivin declined markedly in transfected cells,the ratio of expression product to β-actin in treated group had significant differences compared with negative group and mock group（P<0.01）.The results of FCM indicated that the cells in treated group were inhibited in G₀/G₁ stage,the apoptotic rate was 20.65%±1.28%，there were significant differences compared with mock group and negative group (P<0.01).AO/EB double dyeing showed that the most cells in treated group were in early or middle stage apoptosis.Conclusion The recombinant plasmid of pSU6-si-survivin can significantly inhibit the expression of survivin gene in Bel7402 cells,and suppress cell growth and induce apoptosis，so survivin may act as an important target to treat human hepatocellular carcinoma.

Abstract:Objective To investigate the effect of simvastatin on gene expression of L-type calcium channels(LCCs) of myocardial cells in BALB/c mice infected by coxsackievirus B3(CVB3 ) so as to study the therapeutic effect of simvastatin on viral myocarditis.Methods Sixty male BALB/c mice were divided into five groups randomly(n=12).Mice in viral control group and three groups of oral administration of simvastatin(10,30 and 90 mg•kg^-1)were inoculated intrapritoneally with 0.2 mL of CVB3(Nancy strain).Mice in normal control group were inoculated intrapritoneally with 0.2 mL Eagle’〖KG-*3〗s solution.The heart samples of all the mice were obtained for hispathological study and detection of myocardial LCCs alpha1 subunits mRNA expression by semi-quantitative reverse transcription- polymerase chain reaction (RT-PCR).Results In viral control group,the mononuclear inflamematory infiltrate was focal or diffuse in myocardium of mice,severe hearts revealed a large area of myocardial necrosis.The degree of inflammatory cell infiltrate and area of necrosis were significantly less in simvastatin groups as compared with viral control group.The myocardial LCCs alpha1 subunits mRNA expression by semi -quantitaive RT-PCR in normal control group was much lower than that in viral control group (0.06±0.01 vs 1.37±〖JP〗0.32,P<0.05); the expression levels of myocardial LCCs alpha1 subunits mRNA in simvastatin(10,30 and 90 mg•kg^-1)  groups were 1.14±0.22,0.17±0.04 and 0.11±0.02,respectively,and they were lower than that in viral control group(Ｐ<0.05); In simvastatin(30 and 90 mg•kg^-1) groups,the myocardial LCCs alpha1 subunits mRNA expressions were significantly decreased(P<0.05). Conclusion Simvastatin could reduce the grade of myocardial damage and block virus-induced LCCs alpha1 subunit gene expression in a dose-dependent manner,which might exert a protective effect in myocarditis mice infected by CVB3.

Abstract:Objective To study the treating effect of middle and high doses of acetone and aether extracts from Rumex.Japonicus Houtt on mouse thrombocytopenia.Methods 80 BACB/C mice were randomly divided into 5 groups　(n=16): negative control group(salad oil),middle dose (0.5 g•kg^-1) and high dose(5 g•kg^-1 )of acetone and aether extracts groups.All mice in various group were injected with cyclophos-phamide （0.3 mL/20 g） by hypodermic for 3 d to set up the thrombocytopenic model; after mouse thrombocytopenic model was successfully set up,the mice were given salad oil,middle and high doses of acetone and aether extracts,respectively, by gastric perfusion，20 d later the mice were executed,the numbers of the platelet,red cells and white cells were determined.Results ①The numbers of platelet numbers of all groups on the 14th day after making model decreased significantly(P<0.01)；②When the experiment was finished,the platelet numbers of mice were significantly increased compared with the 14th day(P< 0.01)，the platelet numbers of experimental groups were obviously increased compared with negative control group(P< 0.01)；③ There were no significant differences of the numbers of red cells and white cells between experimental groups and negative control group(P>0.05).Conclusion Middle and high doses of acetone and aether extracts have obviously curative effect on mouse thrombocytopenia.

Abstract:Objective To explore the effect of TIP30 gene on the growth inhibition of renal carcinoma cell line 786-0 and look for a potential therapeutic target for renal carcinoma.Methods TIP30 gene was amplified by reverse transcription-polymerase chain reaction(RT-PCR).A eukaryotic expression vector pcDNA3.1-TIP30 was constructed and transfected into 786-0 cells; pcDNA3.1(+)was also transfected as control.After transfection,the expression of TIP30 in 786-0 cells was detected by RT-PCR and Western blotting.The changes of cell proliferation and cell cycle were observed by MTT and FCM assay.Results The mRNA and protein expressions of TIP30 gene in pcDNA3.1-TIP30-transfected 786-0 cells were significantly increased than those in untreated and pcDNA3.1(+)-transfected cells(P<0.05).However,there was no difference in TIP30 expression between untreated and pcDNA3.1(+)-transfected 786-0 cells(P>0.05).The inhibitory rate of pcDNA3.1-TIP30-transfected 786-0 cells was significantly higher than those in untreated and pcDNA3.1(+)-transfected cells(P<0.01); Cell cycle analysis by flow cytometry showed that the number of cells in G₀-G₁ phase of pcDNA3.1-TIP30-transfected 786-0 cells was significantly increased while the number of cells in phase S and G₂-M was decreased compared with untreated and pcDNA3.1(+)-transfected cells(P<0.01).Conclusion 786-0 cells can stably express exogene TIP30. Transduction of TIP30 gene into lower expression renal carcinoma cells can restore its suppressive effect on cell growth,suggesting that TIP30 gene may be a new therapeutic target for renal carcinoma.

Abstract:Objective To study the expression and location of Na⁺-K⁺-2Cl^-cotransporter (NKCC₂) and gamma epithelial Na⁺ channel(γENaC) in kidney tissues of rats with acute renal failure (ARF) and to explain the pathophysiological mechanism of water and sodium disorders during ARF.Methods The ARF rats were induced with glycerol by intramuscular injection (ARF group),the control rats were administered with the equal volume of normal saline (control group).Then the pathological alteration of kidney tissues and the serum and urine biochemical parameters of rats were examined,and the expressions of NKCC₂ and γENaC and their cellular location in kidney tissues were also determined by Western blotting and immunohistochemistry (IHC).Results The urine volumes (mL•d^-1)(35.2±8.7,47.9±11.0, and 43.2±17.0 on the first,second and third day, respectively)increased after intramuscular injection of glycerol,they were higher than that of control (P<0.05) ,but decreased gradually to the normal level on the fourth day(22.0±5.2). The serum creatinine and blood urea nitrogen levels in ARF rats were (53.8±12.2) μmol•L^-1 and (9.5±1.2) mmol•L^-1 respectively,and they were higher than those in control group (38.9 μmol•L^-1 ±9.1 μmol•L^-1 and 7.9 mmol•L^-1±1.5 mmol•L^-1 respectively) (P<0.05).　The pathological results showed the considerably shrinked renal tubules in diameter,the adipose degeneration of tubular epithelial cells and the immune cell infiltration in the kidney tissues of ARF rats.The increased NKCC₂ and γENaC expressions of ARF rats were found by IHC,and this was further proved with Western blotting,the ratio of membrane NKCC₂ and γENaC expressions in tubular epithelial cells (2.54±0.27 and 1.83±0.23) increased significantly when compared with the ratio of plasma (1.53±0.17 and 1.21±1.18) (P<0.05).Conclusion During ARF period,both membrane and cytoplasm NKCC₂ and γENaC expressions increase,and the increased membrane protein is the key step for sodium reabsorption.And this suggests that the compensatory enhanced function of the partial nephron with normal structure occurs,and the membrane location or the increased level of sodium transporter involves in the metabolism of sodium and water in the diseased kidney.

Abstract:Objective To investigate the genetic assaciation between the estrogen receptor-alpha gene polymorphism and uterine leiomyoma in Northern Han Chinese.Methods The method of PCR-RFLP was conducted to examine the genotypes of rs722209 and rs9322346 of estrogen receptor-alpha gene in 337 subjects,including 167 uterine leiomyoma and 170 controls.The association between the polymorphisms and uterine leiomyoma was analyzed with SPSS 12.0.Results The genotypic frequencies of the 2 SNPs of estrogen receptor-alpha gene did not deviate from Hardy-Weinberg equilibrium in both patient and control groups.The allelic and genotypic frequencies of the 2 SNPs had no remarkable difference between patient and control groups(P>0.05).Conclusion The gene polymorphisms of rs722209 and rs9322346 of estrogen receptor-alpha may be not associated with uterine leiomyoma.

Abstract:Objective To investigate the reversal mechanism of tetrandrine (TTD) on multidrug resistance(MDR) in leukemia cell line K562/A02.Methods Human leukemia cell line K562 and multidrug-resistant cell line K562/A02 were used as the target cells.There were five groups in the examination of cellular level(K562 group,K562/A02 group,K562+ADM group,K562/A02+ADM group and K562/A02+TTD+ADM group）.The non-cytotoxicity doses to cell lines K562 and K562/A02 of TTD were got by MTT assay.Using flow cytometry (FCM) assay to examine the intracellular ADM concentration.There were three groups in the examination of genic,zymologic and protein levels(K562 group,K562/A02 group and K562/A02+TTD group）.The mRNA expression of MDR was measured by fluorescent quantitative reverse transcriptase polymerase chain reaction(RT-PCR).The expression levels of glutathione-S-transferase (GST-π) and topoisomerase Ⅱ(Topo Ⅱ) were determined by immunohistochemical technique.The expressions of P-glyco-protein(P-gp) and bcl-2 were observed by Western-blotting.Results The intracellular concentration of ADM in K562/A02 cells after treated with 1.562　5 mg•L^-1 TTD was higher than that in ADM groups(P<0.01).Compared with blank group,the expression of mdr1 mRNA/ P-gp in K562/A02 cells reduced(P<0.01),the expressins of GST-π and Topo Ⅱ had no change,and the expression of bcl-2 reduced（P<0.01）.Conclusion TTD can reverse the MDR of K562/A02 cell line by inceasing intracellular concentration of ADM and reducing the expressions of mdr1 mRNA/ P-gp and bcl-2 in K562/ A02 cells.

Abstract:Objective To transfect a recombinant pcDNA3.1-DCN into HepG2 cells and detect the expressions of decorin(DCN) and p21^WAF1/CIP1 so as to investigate the mechanism of tumor suppression of DCN.Methods HepG2 cells were divided into pcDNA3.1-dec-HepG2 group(transfected group) and pcDNA3.1-HepG2 group (control group).After the recombinant pcDNA3.1-DCN and pcDNA3.1 were transfected into HepG2 hepatoma cells by liposome,the stably transfected cell lines were established using G418 screening. RT-PCR method was used to detect the expressions of DCN and p21^WAF1/CIP1 mRNA; Western blotting method was used to detect the expressions of DCN and p21^WAF1/CIP1 protein; immunohistochemistry was performed to detect the expression of DCN protein.Results Compared with control group,the expressions of DCN and p21^WAF1/CIP1 mRNA were significantly increased in transfected group by RT-PCR (P<0.05), the expressions of DCN and p21^WAF1/CIP1 protein increased obviously in transfected group by Western blotting (P<0.05),the expression of DCN protein increased in transfected group by immunohistochemistry (P<0.05). Conclusion The stably transfected HepG2 cell lines with DCN is successfully established and it is proved that DCN can increase the mRNA and protein expressions of p21^WAF1/CIP1.

Abstract:Objective To investigate the changes of Bcl-2 and Bax expressions on human hepatocarcinoma SMMC-7721 cells injuried with vitamin K3(VK3) treatment and provide information for further research of its mechanism.Methods SMMC-7721 cells were cultivated in vitro,then were divided into control group and experimental groups treated with different concentrations of VK3（20,40 and 60 μmol•L^-1）.The viability of SMMC-7721 cells treated with VK3 was measured by MTT assay,LDH release rate was detected by ultraviolet spectrophotometry,the expressions of Bcl-2 and Bax mRNA were determined using RT-PCR,the expressions of Bcl-2 and Bax protein were assayed by using Western Blotting.Results Compared with control group,20 μmol•L^-1 VK3 did not induce significant changes of cell viability,LDH release rate,the expressions of Bcl-2 and Bax mRNA,the expressions of Bcl-2 and Bax protein. In 40 and 60 μmol•L^-1VK3 groups the cell viabilities decreased (P<0.05 or P<0.01),the LDH release rates increased (P<0.05 or P<0.01),the expressions of Bcl-2 mRNA decresed (P<0.05),the expressions of Bax mRNA increased(P<0.05),the expressions of Bcl-2 protein decresed,the expression of Bax protein increased compared with control group.Conclusion 40 μmol•L^-1VK3 can induce SMMC-7721 cell injury,its mechanism may be related to down-regulation of Bcl-2 protein expression and up-regulation of Bax protein expression.

Abstract:Objective To investigate the charateristics of pathological changes in hippocampal subregions in rats with epilepsy induced with different doses kainic acid (KA) ,discuss the etiology and pathway of epileptic wave.Methods 30 male Wistar rats were randomly divided into control group,low（0.025 μg） and high（0.1 μg） dose KA injection groups with 10 rats each.KA was focally injected into the right amygdala by a glass micropipette connected to an air pressure system to make epilepsy model.The pathological characteristics in hippocampal subregions in rats with epilepsy induced with different doses KA were observed. Results Compared with control group，high dose KA injection mainly caused neuron loss in the CA3 region,while pyramidal and dentate granule cells were evenly distributed with normal shape and size.Low dose KA injection caused severe damage in both CA1 and CA3 regions.Dentate granule cells didn’ t show any pathological change and neuron loss in low dose injection.Conclusion The pathological changes in hippocampal subregions in rats with epilepsy induced with KA are different with different doses KA,it might be related to the pathway of epileptic wave and the specific properties of hippocampus.

Abstract:Objective To observe the effects of rosiglitazone(RSG) on myocardial infarct size (IS),incidence rate of arrhythmias and the pathohistological changes in rat model of acute myocardial ischemia-reperfusion(I-R) injury.Methods The rat myocardial I-R injury model was induced by ligating left anterior descending branch of coronary artery for 30 min and reperfusing for 120 min.Wistar rats were randomly divided into four groups: I-R group,RSG groups with the doses of 3 and 6 mg•kg^-1,and sham-operation group.IS, times and types of arrhythmia, pathohistological changes of myocardium tissue and changes of myocardial cell ultrastructure were observed.Results Compared　 with I-R group,RSG decreased the ratio of IS/LV%（29.3±4.9 vs 37.6±3.2,P<0.05） and the rate of IS/AAR%(76.1±9.6 vs 93.5±7.4，P<0.05),reduced the score of arrhythmias（2.6±0.4 vs　46.2±0.6，P<0.05),the injuries of pathohistology of myocardium and myocardial cell ultrastructure in RSG group were better that those in I-R group.Conclusion RSG has the protective effect on acute myocardial I-R injury in rats，probably by reducing myocardium ischemia area and incidence rate of arrhythmias and ameliorating morphology and ultrastructure of myocardium.

Abstract:Objective To investigate the best time of the mesenchymal stem cells (MSCs) transplantation after myocardial infarction (MI),in order to provide prophase experimental basis for treatment of the diseases correlating to cadiocyte necrosis by cells transplantation. Methods Thirty New Zealand big-ears albino rabbits,which aging were older than or equal to 36 months were selected(a 36-month rabbit is the advanced age rabbit,equaling a person 60 years old).Model of MI was made by ligating anterior descending branch.The model rabbits were randomly divided into six groups(control group,1 d group,1-week group,2-week group,3-week group; and 4-week group,n=5).200 μL cells after marked were drawed and injected in multiple positions of myocardium after MI in 1 d group,1-week group,2-week group,3-week group and 4-week group separately.The rabbits in control group were injected with saline of equal quantity.The cardiac functional parameters,blood flow rate of atrioventricular valve and the hemodynamics at eighth week in various groups were measured.Results Compared with control group,the treatment of MSCs transplantation for MI improved the heart function.The cardiac functional parameters,blood flow rate of atrioventricular valve and the hemodynamics in 1 d group was higher than those in control group （P<0.05）;the cardiac functional parameters,blood flow rate of atrioventricular valve and the hemodynamics in 1-week group,2-week group,and 3-week group were significantly higher than those in control group （P<0.01）; there was no significant difference between 4-week group and control group (P>0.05) .The cardiac functional parameters,blood flow rate of atrioventricular valve and the hemodynamics in 1-week,2-weeks and 3-weeks groups were better than those in 1 d group (P<0.05),however there was no significant difference between them(P>0.05).Conclusion In the condition of the same cell-density but the different time of MI,the transplantation time window is 1 d-3 weeks after MI,which is advantageous to the amelioration of heart function,the best time of transplantation was 1-3 weeks after MI.

Abstract:Objective To investigate the inhibitory effects of Juglans Mandshurica Maxim stem-bark extract（JMME） on SMMC-7721，MCF-7 and A549 cells in vitro,and to confirm the antitumor medicative value of JMME, and to verify antitumor mechanisms of JMME by examining the apoptosis and telomerase activitiy change induced by JMME.Methods SMMC-7721， MCF-7 and A549 cells in culture medium were treated with 25,50,100,200,400 and 800 mg•L^-1 JMME respectively.The inhibitory rates of these cells were measured by MTT assay.After SMMC-7721,MCF-7 and A549 cells in culture medium were respectively treated with 200 mg•L^-1 JMME,the morphological changes of cells were observed under inverted microscope and the apoptosis was detected by agarose gel electrophoresis assay.Then PCR-enzyme-linked immunosorbent assay(ELISA) was used to detect telomerase activities of SMMC-7721,MCF-7 and A549 cells.Results The inhibitory rates (%) of JMME with different concentrations（25,50,100,200,400 and 800 mg•L^-1） on SMMC-7721 were 13.06，21.77，29.88，41.74，69.82 and 78.08，respectively； 24.90，35.61，45.42，65.65，69.69 and 81.39 on MCF-7； 12.32，26.21，41.65，55.38，62.87 and 80.13 on A549.The results showed that JMME had cytolytic action on tumor cells compared with control group（P<0.05）.The inhibitory rates increased with the increasing of the concentration of JMME(P<0.05）.IC₅₀ of SMMC-7721,MCF-7 and A549 after treated with JMME were 211.21,111.07 and 176.20 mg•L^-1,respectively.The IC₅₀ of MCF-7 cells were lower than those of SMMC-7721 and A549 cells.The typical apoptosis morphology was identified under inverted microscope.The typical ladder bar was observed.These results demonstrated that the apoptosis of tumor cells were induced by JMME.The telomerase activities of SMMC-7721,MCF-7 and A549 were stronger than that in negative control ，but the telomerase activities of the tumor cells treated with extracts were much weaker than that of the tumor cells untreated.The inhibitory rates were 42.86%,73.30% and 58.78%,respectively.Conclusion JMME has antitumor effect in vitro.It’ s mechanism is associated with inducing tumor cells apoptosis and inhibiting telomerase activity.

Abstract:Objective To observe the effect of ionizing radiation on the expressions of TLR4 and adaptor protein MyD88 in mouse peritoneal macrophages in the Toll-mediated signal transduction pathways. Methods 160 male Kunming white mice were randomly divided into 16 groups ( ten each group )：sham-irradiation and five groups after 4,8,16,24 and 48 h X-ray irradiation for time-course experiments;sham-irradiation and nine groups after 0.05,0.075,0.100,0.200,0.500,1.000,2.000,4.000,6.000 Gy irradiation for dose-effect experiments. Flow cytometry was used to detect the expressions of TLR4 and MyD88 after whole body irradiation (WBI) with X-rays. Results Both of TLR4 and Myd88 expressed more than sham-irradiation after 0.075 and 2.000 Gy WBI for 4 h，and the expressions of them reached the peak at 16 h or 4 h after WBI(P<0.05 or P<0.01) in time-course experiments. In dose-effect experiments,the expression levels of TLR4 and MyD88 increased gradually with the enhancement of X-rays irradiation dose，both of them reached the peak at 2.000 Gy WBI,and there was significant difference compared with sham-irradiation group (P<0.01). Conclusion X-ray irradiation can increase the expressions of TLR4 and MyD88 in mouse peritoneal macrophages，promote the immune respose and start the adaptive immunity of mice.

Abstract:Objective To explore the protective effects of Ginkgo biloba extract （GBE）on ECV304 cells exposed to high-level glucose and its mechanisms . Methods ECV304 cells were divided into four groups:high-level glucose group,GBE protective group,mannitol high-osmosis group and normal group. In vitro,ECV 304 cells were exposed to 35 mmol•L^-1 glucose for 24 h. The cells from different groups were collected,the production of super anion(O^-₂), hydroxyl radical(•OH)，level of malonaldehyde ( MDA),and activity of superoxide dismutase(SOD) were determined. The structures of mitochondria of cells in different groups were also observed under electronic microscope. Results The level of MDA and the production of O^-₂,•OH in ECV304 cells in high-level glucose group were significantly higher than those in GBE group（P<0.05）,the SOD activity was significantly lower than that in GBE group（P<0.05）. The level of MDA and production of O^-₂，•OH in mannitol group were lower than those in high-level glucose group(P<0.05).Under electronic microscope,the mitochondrial structure of ECV 304 cells in GBE group was normal and cristea were clear and in perfect shape,but the mitochondrial structure of ECV 304 cells in high-level glucose group was swollen and cristea were disappeared. Conclusion GBE plays a protective role in ECV 304 cells exposed to high-level glucose.

Abstract:Objective To investigate the gene transduction efficiency of lentiviral vector in leukemia cells to provide key basis for leukemia gene therapy. Methods A third-generation self-inactivating (SIN) lentiviral vector system based on human immunodeficiency virus type 1 (HIV-1) was used to improve transduction efficiency. The transduction efficiency of the HIV-1-based vector was compared directly with the moloney murine leukemia virus (MLV) SIN vector in human leukemia cell line K562. The expression of green fluorescent protein(GFP) in cells was observed by fluorescence microscopy and flow cytometry (FCM) to detect the percentage of gene trasduction cells. Results The GFP expression in K562 cells was observed qualitatively by fluorescence microscopy. At the same gene transduction conditions,the GFP marker gene expression intensity and GFP positive cells in leukemia cells transduced with HIV vectors were significantly higher than those transduced with MLV vectors. Initial transduction efficiencies were almost 100% for the HIV and less than 40% for the MLV vectors. The transduction efficiency had significant difference between HIV vector group and MLV group（P＜0.05）. Conclusion This lentiviral vector is an excellent gene transduction system for leukemia cells because of its high gene transduction efficiency.

Abstract:Objective To obtain the calcinated bone ceramics used for bone defects repair with a good biological compatibility and bone guidance function,and study its physicochemical characteristics in order to find ideal stent for bone defects repair. Methods The ox bone was calcinated to remove the organic principle,the antigenicity and the pathogenic microorganisms. The inorganic principle which was same to the human bone was retained to maintain the crude 3D communication structure. After pretreatment the ox bone was calcinated to 850℃ and became the granular. The physicochemical characteristics and chemical composition were determined by X-ray diffraction,electron microprobe,inductively coupled plasma atomic spectrometry,electron microscopy and photoelectron spectrometry. Results Hydroxyapatite(HAP) was the main composition of the calcinated bone ceramics determined by X-ray diffraction. No nitrogen was detected by inductively coupled plasma atomic spectrometry. This result showed that there was not protein and organic principles in the calcinated bone ceramics. The crude bone trabecular,trabecular bone cavity and space system were observed clearly by scanning electron microscope. The pore size was 200~850 μm. The average porosity was 84.9%±0.6%. Conclusion There are 3D communication pore structure in calcinated bone ceramics.The main composition of calcinated bone ceramics is hydroxyapatite.It has good tissue compatibility.So calcinate bone ceramics is an ideal material for bone repair.

Abstract:Objective To explore the molecular mechanism of TGFβ in the progress of breast cancer by examining the expressions of TGFβ₁,TGFβ receptor Ⅱ and Smad transcriptional factors in normal human breast epithelium cells and MCF7 cells. Methods RT-PCR was used to measure the expression levels of TGFβ₁,TGFβ receptor Ⅱ,Smad4,Smad6 and Smad7 in normal human breast epithelium cells and MCF7 cells. Results There were no any significant differences of the ratios of TGFβ₁,TGFβ receptor Ⅱ,Smad6,Smad7 to GAPDH between normal human breast epithelium cells （0.721±0.214,1.001±0.312,0.689±0.12,0.221±0.124）and MCF7 cells （0.698±0.324,0.998±0.217,0.718±0.257,0.246±0.138）while the expression of Smad4 in MCF7 cells was significantly lower than that in normal human breast epithelium cells (P<0.05). Conclusion The low expression of Smad4 in MCF7 cells may be associated with the promotion effect of TGFβ on breast cancer.

Abstract:Objective To explore the expression of cell death regulatory gene GRIM-19 in human renal clear cell carcinoma tissues and normal renal tissues and significance,and clarity the effect of GRIM-19 in the process of renal clear cell carcinoma.Methods The expressions of GRIM-19 were studied by means of immunohistochemistry in 21 renal clear cell carcinoma tissues and 7 normal renal tissues. RT-PCR was used to detect the mRNA levels of GRIM-19 in 4 normal renal tissues and 8 renal clear cell carcinoma tissues. Results The results of immunohistochemistry indicated that the positive rates of GRIM-19 were 52.3% （11/21）and 100% （7/7） in renal clear cell carcinoma tissues and normal renal tissues,the difference was significant(P<0.05) . The positive rates of GRIM-19 were 100%（6/6），40%（4/10）and 20%（1/5）in the grade Ⅰ,Ⅱand Ⅲ of renal clear cell carcinoma,the differences between them were significant(P<0.01).RT-PCR results indicated that the positive rates of GRIM-19 were 62.5% （5/8）and 100% （4/4）in renal clear cell carcinoma tissues and normal renal tissues,the difference between them was significant（P<0.01）.Conclusion The expression of GRIM-19 obviously decrease in renal clear cell carcinoma tissue in the levels of gene and protein and the decreasing exists relevance with the grade of pathology. It indicates that GRIM-19 expression may be play an important role in the progress of renal clear cell carcinoma.

Abstract:Objective To investigate the association between SAPAP3 gene polymorphism and obsessive compulsive disorder　and provide theoretical basis for its susceptibility gene study. Methods The method of PCR-RFLP was conducted to examine the genotypes of a tag SNP rs11264155 locus on SAPAP3 gene in 190 obsessive compulsive disorder undergraduates (case group) and 190 healthy undergraduates (control group). Results The fragment of rs11264155 was 219 bp,and three genotypes (CC,CG,GG)were found after digestion. The distribution of genotypic frequency of rs11264155 was consistent with Hardy-Weinberg equilibrium. The frequencies of allele and genotype of rs11264155 showed no significant difference between case and control groups(P>0.05). In different clinical types of obsessive compulsive disorder,the frequencies of allele and genotype of rs11264155 showed no significant difference (P>0.05). Conclusion The tag SNP rs11264155 of SAPAP3 gene may not be associated with obsessive compulsive disorder.

Abstract:Objective To detect the expressions of somatostatin receptor subtypes SSTR2 and SSTR5 mRNA in patients with hepatocellular carcinoma (HCC) and investigate the relationship between expression rates and carcinoma characters,in order to provide theoretical foundation for diagnosis and therapy of HCC. Methods 22　 patients with HCC were included,all the specimens with HCC were identified by surgery and the histopathological examination. The expressions of SSTR2 and SSTR5 mRNA in carcinoma tissues,adjacent tissues,and normal tissues were determined and identified with RT-PCR.The relationship between the expression rate and carcinoma characters was analyzed. Results The expression rate of SSTR2 mRNA in carcinoma tissues was 81.8%,the expression level was 118.27±17.25,they were obviously higher than those in normal tissues(36.4%，69.49±19.43) (P<0.05);but there was no difference between carcinona tissues and adjacent tissues(72.7%，111.18±23.74) (P> 0.05);there was obvious difference between normal tissues and adjacent tissues(P<0.05).There was no significant difference of the SSTR5 mRNA expression rates and expression levels between carcinoma tissues(18.2%， 51.67±30.32),adjacent tissues (18.2%，49.56±27.61) and normal tissues(9.1%，48.73±15.72)(P> 0.05). The expression rates of SSTR2 mRNA and SSTR5 mRNA in high differentiation HCC tissues(100% and 25%) and lymph node metastasis negtive HCC tissues(25.0% and SSTR5 100%)were higher than those in low differentiation HCC tissues(0 and 0) and lymph node metastasis positive HCC tissues(0 and 0)（P<0.05）；there was no obvious difference of 　the expression rates between TNM stageⅠ+ⅡHCC tissues(SSTR2 85.7%，SSTR5 21.4%) and TNM stage Ⅲ+Ⅳ(75.0%，12.5%)（P>0.05）. Conclusion The expression level of SSTR2 mRNA in HCC is high.The expression rate and expression level of SSTR2 have close relationship with the differentiantion,lymph node metastsis,and have no correlation with HCC classification.

Abstract:Objective To investigate the variations of hepatic artery and its extrahepatic arteries on hepatic arteriogram and to provide benefit for transhepatic arterical chemoemblization. Methods The hepatic arteriograms of 200 cases with unresectable hepatic malignant tumor before interventional therapy were analysed. Two interventional radiologists in common reviewed the incidences of various types according to Michels’ classification,the absence of proper hepatic artery,and the variations of extrahepatic arteries originating from hepatic artery. Results The most common hepatic artery variation was Michels type Ⅲ(n=17,8.5%),followed by type Ⅱ(n=10,5.0%)　 and Ⅴ(n=9,4.5%). Proper hepatic absence was found in 25 cases and appeared as 5 subtypes. 5 kinds of extrahepatic arteries were found. The most common extrahepatic artery was the right gastric artery (n=156,78.0%),followed by cystic artery (n=126,63.0％),accessory left gastric artery (n=19,9.5％),the hepatic falciform artery (n=5,2.5%),and accessory left inferior phrenic artery (n=4,2.0%). Conclusion There　 are some other variations of hepatic artery beside Michels’ classification,and there are many variations of extrahepatic arteries originating from hepatic artery, it is important to assure interventional therapy effect for hepatic cancer and prevent complication.

Abstract:Objective To investigate the diagnostic value of serum copper and non-ceruloplasmin-bound copper determination in patients with Wilson disease (WD). Methods Ceruloplasmin level was measured by scattering immunoturbidimetric assay and serum copper concentration by flame atomic absorption spectroscopy at the diagnosis of patients with fulminant Wilson disease (FWD,8),non-fulminant Wilson disease (NFWD,49),viral hepatitis （chronic hepatitis 22，severe hepatitis 46,Cirrhosis 19），and other liver diseases (57). Results ①The average serum copper concentration of patients with FWD was ( 105.0±29.4) μg•dL^-1,which was significantly higher than that of patients with NFWD （28.1 μg•dL^-1±14.9 μg•dL^-1) and severe hepatitis （70.5 μg•dL^-1±41.7　μg•dL^-1)（P<0.05）. The serum copper concentration of patients with NFWD was lower than the upper limit of normal,which was significantly lower than those in other groups（P<0.01）. The sensitivity of serum copper concentration was 86.0% for the diagnosis of WD,and the specificity was 47.2%. ② The average serum non-ceruloplasmin-bound copper concentration of patients with FWD was (62.3±27.7) μg•dL^-1 with maximum 113 μg•dL^-1,which was significantly higher than those of other groups（P<0.01）. The average serum non-ceruloplasmin-bound copper concentration of patients with NFWD was (10.2±9.3) μg•dL^-1,which was significantly higher than those of patients with other liver diseases（P<0.01）. The sensitivity of serum non-ceruloplasmin-bound copper concentration was 59.6% for the diagnosis of WD with specificity 100%. Conclusion The concentrations of serum copper and serum non-ceruloplamin-bound copper are valuable in the diagnosis of WD,and the serum non-ceruloplamin-bound copper is a very useful parameter for the diagnosis and prognosis of FWD.

Abstract:Objective To evaluate the malignancy and invasion of gliomas preoperatively by diffusion tensor imaging(DTI),and to study the relationship between gliomas and fiber tracts by diffusion tensor tractography(DTT). Methods A total of 31 patients with grade Ⅰto Ⅳ gliomas（15 cases of gradeⅠand grade Ⅱ gliomas,16 cases of grade Ⅲ and grade Ⅳ gliomas）underwent the conventional MRI and DTI. FA map and DEC map were writen and DTT map was reconstructed in order to make comparison of the rFA values between low-grade gliomas and high-grade gliomas in relative regions of interest (ROIs),as well as to observe the changes of the white fiber tracts. Results A decreasing tendency of rFA values was observed as follow:solid portion lowest (0.428±0.078),followed by edema area(0.578±0.120) and edge of edema(0.834±0.074) in low-grade gliomas;as well as solid protion lowest(0.366±0.055)，followed by edema area(0.458±0.158) and edge of edema(0.6766±0.138) in high-grade gliomas. The rFA values of high-grade gliomas were significantly lower than those of low-grade gliomas in all ROIs(P<0.05). All gliomas presented low signals differently on FA map.The tumor region showed ambiguity and disorder on DEC map. On DTT map,3 of 15 low-grade gliomas showed deviation;the other 12 cases showed slight rarefaction;13 of 16 high-grade gliomas showed significant destruction and partly discontinuation;2 cases showed termination or interruption;1 case mainly showed deviation and part of fiber tracts showed destruction. Conclusion DTI contributes to study malignancy and invasion of gliomas and provide more useful fiber information of gliomas preoperatively.