人端粒酶逆转录酶干扰对肿瘤坏死因子相关凋亡诱导配体诱导肝癌细胞凋亡的影响

目的研究人端粒酶逆转录酶(hTERT)干扰对肝癌．HepG2、SMMC-7221细胞生物学形为的影响和对肿瘤坏死因子相关的凋亡诱导配体(TRAIL)诱导凋亡的影响。方法将HepG2细胞和SMMC-7721细胞分为转染组 (转染重组质粒真核表达载体)、对照组(转染空载体质粒)和未转染组。采用聚合酶链反应方法检测hTERT干扰序列, 逆转录聚合酶链反应方法检测hTERT表达,HE染色、生长曲线和流式细胞术方法分别检测细胞形态、增殖情况和细胞周期,β-半乳糖苷酶染色方法检测细胞状态,Armexin V／PI染色流式细胞术检测细胞凋亡。结果转染组细胞内均存在hTERT干扰序列,HepG2和SMMC 7221细胞hTERT干扰率分别为100％和43．3％;与未转染组细胞相比, 转染细胞核质比明显缩小,增殖率下降差异有统计学意义(P<0．05),老化细胞和G2-M期细胞明显增加(P<0．05)。细胞老化率分别由未转染组的0增加到转染组的20．4％,由3．60%,增加到10．O％;G2-M期分别由未转染组的7．1％、6．9％增加到转染组的10．6％、7．9％。hTERT干扰显著增加肝癌细胞凋亡和TRAIL诱导凋亡敏感性(P<0．05)。两株肝癌细胞凋亡率分别由未转染组的3．5％、4．8％增至转染组的5．2%、7．9％;100 ng／ml TRAIL作用24 h后两株肝癌细胞凋亡率分别由未转染组的5．3％、13．9％增加到转染组的10．4％、77．2％,而对照组细胞各指标均无显著变化。结论 hTERT干扰明显影响肝癌细胞的生物学行为,显著增加细胞凋亡和TRAIL诱导凋亡的敏感性。     

人端粒酶逆转录酶干扰增强肿瘤坏死因子相关的凋亡诱导配体诱导肝癌细胞凋亡的机制

目的探讨人端粒酶逆转录酶(hTERT)干扰增加肿瘤坏死因子相关的凋亡诱导配体(TRAIL)诱导肝癌HepG2和SMMC 7721细胞凋亡的分子机制。方法采用膜联蛋白V/碘化丙锭染色的流式细胞术方法检测细胞凋亡;采用Western blot方法检测凋亡相关蛋白Procaspase-8、9、-3及Bax、Bcl-2和hTERT表达;采用端粒重复扩增法和端粒数量和长度测定法检测端粒酶活陛和端粒长度。结果hTERT干扰显著增加TRAIL诱导的肝癌细胞凋亡。100 ng/ml TRAIL作用24 h后,HepG2细胞凋亡率由5.53%增加至10.35%;SMMC 7721细胞凋亡率由14.73%增加至77.24%。hTERT干扰明显增加Procaspase-8、-9和Bcl-2表达,显著降低Bax表达,明显促进TRAIL作用后Procaspase-8、-9、-3活化,并且hTERT干扰后端粒酶活性显著降低,端粒长度明显缩短,然而对照细胞与未转染细胞相比各指标均无明显变化。结论hTERT干扰明显增加TRAIL诱导的肝癌细胞凋亡,其机制可能与Procaspase-8、-9表达增加,端粒酶活性降低和端粒长度缩短有关,而与Bcl-2和Bax表达无关。     

全反式维甲酸联合奥沙利铂对人SMMC-7721肝癌细胞株的作用

目的:观察全反式维甲酸(ATRA)联合奥沙利铂(L-OHP)对人SMMC-7721肝癌细胞增殖及凋亡的影响.方法:选用ATRA(10-5mol/L)及不同浓度的L-OHP(10mg/L、20mg/L、40mg/L),并选用10-5mol/L的ATRA分别联合L-OHP(10mg/L、20mg/L、40mg/L)作用于SMMC-7721肝癌细胞24h、48h、72h;采用MTT比色法观察其对SMMC-7721肝癌细胞的生长抑制作用,倒置显微镜下观察细胞形态变化;采用流式细胞术分析药物作用48h时SMMC-7721细胞的周期分布和凋亡的情况.结果:ATRA及不同浓度L-OHP单药及联合均可显著抑制SMMC-7721肝癌细胞的生长,细胞形态改变,凋亡比例增加,并呈剂量-时间依赖性;两药联合较单药相比作用明显增强(P<0.01);ATRA联合20mg/LL-OHP与ATRA联合40mg/LL-OHP相比,作用细胞48h及72h时,对细胞生长抑制作用无统计学差异;两药联合较单药相比,细胞凋亡率明显增加,细胞阻滞S期增强(P<0.01).结论:ATRA可抑制肝癌SMMC-7721细胞的生长并诱导细胞凋亡,与L-OHP联用后作用增强并可减少奥沙利铂用量,具有协同作用.     

Apoptosis of hepatoma cells SMMC—7721 induced by Ginkgo biloba seed polysaccharide

AIM：To study the apoptosis of hepatoma cells SMMC-7721induced by polysaccharide isolated from Ginkgo biloba seed.METHODS:Ginkgo biloba seed polysaccharide(GBSP)was isolated by ethanol fractionation of Ginkgo biloba seed and purified by Sephadex G-200 chromatography,The purity of GBSP was verified by reaction with iodine-potassium iodide and ninhydrin and confirmed by UV spectrophotometer,cellulose acetate membrane electrophoresis and Separose 4B gel filtration chromatography,The Scanning Electron Microscope(SEM)and Flow Cytometry(FCM)were used to examine the SMMC-7721 cells with and without GBSP treatment at 500mg/ml for 36h.RESULTS:GBSP product obtained was of high purity with the average molecular weight of 1.86&#215;10^5.Quantitative analysis of SMMC-7721cells in vitro with FCM showed that the percentages of G2-Mcells without and with GBSP treatment were17.01&#177;1.28%and 11.77&#177;1.50%(P&lt;0.05).the debris ratio of the cells were0.46&#177;0.12%and 0.06&#177;0.06%(P&lt;0.01).and the apoptosis ration of cells was3.84&#177;0.55%and9.13&#177;1.48%(P&lt;0.010respectively,Following GBSP treatment,microvilli of SMMC-7721cells appeared thinner and the number of spherical cells increased markedly,Most significantly,the apoptosis bodies were formed on and around the sppherical cells treated with GBSP.CONCLUSION:GBSP could potentially induce the apoptosis of SMMC-7721cells.     

Norcantharidin combined with ABT-737 for hepatocellular carcinoma: Therapeutic effects and molecular mechanisms

AIM: To study the therapeutic effect of norcantharidin(NCTD) combined with ABT-737 on hepatocellular carcinoma cells and the molecular mechanism. METHODS: Two hepatocellular carcinoma(HCC) cell lines, Hep G2 and SMMC-7721, were selected. ABT-737 and NCTD were allocated into groups to be used alone or in combination. Hep G2 and SMMC-7721 cells were cultured in vitro. Liver cancer cells in the logarithmic phase of growth were vaccinated and cultured to the cell wall stage; these cells were treated for 48 h with different concentrations of NCTD, or ABT-737, or NCTD combined with ABT-737. The cell proliferation inhibition rate was detected by methyl thiazolyl tetrazolium. The expression of Mcl in HCC cells was detected by Western Blotting, and the cells in each group after treatment had apoptosis detected by flow cytometry. The proliferation inhibition rate, the expression of Mcl-1 in cells and the apoptosis inducing effect of treatment were observed in each group, and the effect of NCTD on ABT-737 in the treatment of HCC and its mechanism of action were analyzed.RESULTS: As the concentration of NCTD increased, the cell proliferation inhibition rate gradually decreased; and the treatment effect of ABT-737 1-3 μm combined with NCTD on cell proliferation inhibition was stronger than that of ABT-737 alone. The difference was statistically significant(P 0.05). In observing the expression of Mcl-1 in cells after the treatment of different concentrations of NCTD, this was partiallyinhibited after treatment with NCTD 15 μm, and the expression of Mcl-1 was almost undetectable after treatment with NCTD 30 μm and 60 μm. The effect on inducing apoptosis with the treatment of ABT-737 or NCTD alone for 48 h was lower than that of the control group. The difference was not statistically significant(P 0.05). The effect on inducing apoptosis in Hep G2 and SMMC-7721 cells with the treatment of ABT-737 combined with NCTD for 48 h was greater than that of ABT-737 or NCTD alone. The difference was statistically significant(P 0.05). CONCLUSION: NCTD combined with ABT-737 has a positive role in the treatment of HCC, and it has great value in clinical research.     

人端粒酶催化亚单位锤头状核酶诱导肝癌细胞凋亡的作用

目的 构建带有U6启动子的人端粒酶催化亚单位锤头状核酶真核表达质粒及其突变体,转染入肝癌细胞株SMMC7721,观察端粒酶活性、细胞增殖和凋亡的情况。 方法 用分子克隆技术构建由U6作为启动子、绿色荧光蛋白基因作为报告基因的核酶真核表达质粒pGTRz-U6及其突变体pGTmRz-U6,并以空质粒pEGFP-C1作为对照。Lipofectamine2000转染人肝癌细胞株SMMC7721,G418筛选阳性克隆。RT-PCR检测核酶及hTERT基因的表达,四甲基偶氮唑盐(MTT)作细胞生长曲线观察其生长情况,TRAP-银染法检测端粒酶活性变化,流式细胞计数(FCM)法检测细胞的凋亡水平。 结果 核酶、突变核酶在SMMC7721中持续表达;凝胶成像系统分析SMMC7721-pEGFP-C1、SMMC7721-mRz、SMMC7721-Rz hTERT基因表达,用SPSS10.0软件对3种细胞进行分析,发现三者hTERT基因表达水平不同(F=47.987,P<0.01);t检验分析得出SMMC7721-Rz hTERT基因表达明显低于SMMC7721-mRz和SMMC7721- pEGFP-C1(t值分别为-7.640和-11.602,P值均<0.01)。SMMC7721-pEGFP-C1和SMMC7721-mRz hTERT表达没有区别(t=-0.178,P>0.05)。TRAP-银染及FCM结果分别显示,随着细胞的分裂,SMMC7721-Rz和SMMC7721-mRz细胞端粒酶活性逐渐降低,凋亡水平逐渐增加,7PDS细胞凋亡率分别是29.86％和9.87％,而对照组SMMC     

Bcl-2核酶对SMMC7721细胞的促凋亡机制

目的 观察Bel-2核酶对SMMC-7721细胞的作用,探讨bcl-2抑制细胞凋亡的机制。方法 经脂质体介导的方法将PMTr-neo(正向Bcl-2核酶真核表达载体)导入SMMC 7721细胞中．细胞克隆转移扩大培养后,采用TUNEL,TRAP结合ELISA流式细胞仪,免疫组化技术检测SMMC 7721／PMTr-neo细胞增殖及细胞凋亡。结果 较对照组SMMC 7721／PMTr-neo细胞bcl-2表达水平显著下降,伴有显著细胞凋亡现象及端粒酶活性下降,结论 Bcl-2核酶可促进SMMC 7721细胞发生凋亡,并降低细胞端粒酶活性,为反义技术在肝癌治疗中应用提供理论依据。     

端粒酶逆转录酶基因反义寡核苷酸抑制肺癌细胞端粒酶活性和诱导细胞凋亡

目的 研究端粒酶逆转录酶（hTERT)基因反义寡核苷酸（ASODN）对肺癌细胞端粒酶活性和细胞凋亡的影响。方法 实验分为ASODN组、正义寡核苷酸（SODN）组和空白对照组,所用ASODN和SODN的浓度分别为10μmol/L,脂质体为16mg/L,采用端粒重复扩增法、逆转录－聚合酶链反应、Western Blot及流式细胞术分别观察各组端粒酶活性、hTERP mRNA和蛋白质表达以及细胞凋亡。结果 ASODN组显著下调或抑制肺癌细胞端粒酶活性和hTERT表达,但直到第21天才出现细胞凋亡增多。结论 端粒酶活性与hTERT表达密切相关。     

Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.     

Effect of Nimesulide on proliferation and apoptosis of human hepatoma SMMC—7721 cells

AIM:Cyclooxygenase-2(COX-2)has been suggested to be associated with carcinogenesis.We sought to investigate the effect of the selective COX-2 inhibitor,Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS:This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line Various concentrations of Nimesulide(0,200μmol/L,300μmol/L,400μmol/L)were added and incubated.Cell proliferation was detected with MTT colorimetric assay,cell proliferation was detected with MTT colorimetric assay,cell apoptosis by electron microscopy,flow cytometry and TUNEL.RESULTS:Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the controla group.The duration lowerst inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%,the highest inhibition rate was 58.49%,After incubation with Nimesulide for 72h,the most highest apotosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%&#177;1.62%,vs2.24%&#177;0.26%and 21.23&#177;1.78vs2.01&#177;0.23(P&lt;0.05).CONCLUSION:The selective COX-2 inhibitor,Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells,The apoptosis rate and the apoptosis index are dose-dependent.Under electron microscope SMMC-7721 cells incubated with 300 μmol and 400μmol Nimesulide show apoptotic characteristics With the clarification of the mechanism of selective COX-2 inhibitors,Thtese COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.     

羟基喜树碱诱导SMMC-7721细胞凋亡时线粒体凋亡诱导因子的转位研究

目的研究羟基喜树碱诱导肝癌细胞株SMMC-7721凋亡时线粒体凋亡相关蛋白凋亡诱导因子表达及从线粒体发生核转位的变化.方法用80 μg/ml羟基喜树碱作用于肝癌细胞株SMMC-7721后,用吖啶橙/溴化乙啶染色法观察细胞凋亡现象;用电子显微镜观察线粒体超微结构;分别用逆转录聚合酶链反应、Western blot检测凋亡诱导因子在mRNA与蛋白质水平表达的变化;用激光共聚焦显微镜观察凋亡诱导因子在细胞凋亡时从线粒体到核的迁移变化.结果 80μg/ml羟基喜树碱作用SMMC-7721细胞后,吖啶橙/溴化乙啶荧光双重染色可见细胞体积缩小、细胞皱缩、核碎裂等典型细胞凋亡形态学改变;超微结构观察发现线粒体肿胀;细胞凋亡时凋亡诱导因子在mRNA与蛋白质水平上的表达与对照组细胞相比没有明显变化,但凋亡诱导因子发生了从线粒体到核的迁移. 结论羟基喜树碱可以通过线粒体途径诱导人肝癌细胞发生凋亡;在线粒体凋亡途径中,线粒体凋亡相关蛋白凋亡诱导因子从线粒体释放并发生核转位可能与羟基喜树碱诱导细胞凋亡密切相关.