Survivin对人胆管癌细胞凋亡相关信号通路的作用机制

目的:探讨Survivin基因对人胆管癌细胞凋亡信号通路的调节机制.方法:构建针对Survivin基因的siRNA和对照siRNA,分别转染QBC939人胆管癌细胞,Western blot检测siRNA对细胞Survivin的干扰效果.继而分别用流式细胞仪,激酶活性测定和Westernblot检测不同Survivin表达状态下,QBC939细胞的凋亡状态,caspase-3的活性和caspase-3,caspase-9及procaspase-9凋亡信号分子的表达.结果:siRNA-Survivin显著抑制Survivin在QBC939细胞的表达(P<0.05).Survivin表达抑制后,QBC939细胞凋亡明显增加(18.9%±2.3%,P<0.05),caspase-3活性显著升高(0.83±0.15,P<0.01),caspase-3和caspase-9表达明显上调(P<0.05),而procaspase-9表达降低(P<0.05).未转染和转染对照siRNA的QBC939细胞上述变化无显著性差异(P>0.05).结论:Survivin基因通过促进procaspase-9的活化以阻止caspase-3和caspase-9的激活从而抑制胆管癌细胞的凋亡.     

shRNA干扰STAT3基因表达对胃癌细胞MKN-45体内外生物学特性的影响

目的:研究shRNA干扰STAT3基因表达对胃癌细胞MKN-45体内外生物学特性的作用.方法:应用本实验室已构建并鉴定的STAT3基因的特异性小RNA干扰质粒(psiRNA-H1/STAT3),使用脂质体转染MKN-45细胞,实验分为对照组、psiRNA-H1转染组和psiRNA-H1/STAT3转染组3组.通过RT-PCR和Western blot检测STAT3特异性小RNA干扰基因对胃癌细胞STAT3基因mRNA和蛋白表达的影响;MTT比色法检测细胞的生长抑制率;流式细胞仪分析MKN-45细胞周期分布.用各组MKN-45细胞建立移植瘤裸鼠模型,筛选转染psiRNA-H1和psiRNA-H1/STAT3的MKN-45稳定细胞株观察裸鼠移植瘤的生长情况.结果: 成功转染重组体的MKN-45细胞中STAT3 mRNA和蛋白表达明显下降(0.612±0.074 vs 1.937±0.043,P<0.05;0.668±0.054 vs 2.005±0.064,P<0.01).G0/G=期细胞比例增高,另一方面S期细胞比例降低,细胞的增殖系数明显降低(25.42±3.48 vs 33.54±2.91,P<0.05).移植瘤裸鼠模型显示,psiRNA-H1/STAT3组瘤体在体积上明显减小(4.47 cm3±0.18 cm3 vs 13.65 cm3±5.64 cm3,P<0.05).结论:针对STAT3基因的特异性小RNA干扰质粒在体外能有效抑制人胃癌细胞系STAT3 mRNA和蛋白表达,细胞增殖能力减弱,在体内明显抑制肿瘤生长,为STAT3基因靶向治疗提供一定的实验依据.     

pEGFP-C1-反义生存素重组质粒的构建和转染

目的 构建pEGFP-C1-反义生存素（survivin)重组质粒，并转染入人MKN-45低分化胃腺癌细胞株，观察转染前后生存素基因的表达及对凋亡的影响。从基因水平探讨反义生存素核酸治疗胃癌的分子生物学机制。为今后利用反义基因治疗肿瘤提供实验和理论依据。方法 应用基因重组技术构建 pEGFP-C1-反义生存素重组质粒，通过脂质体转染法转染人MKN-45胃腺癌细胞株，以流式细胞仪方法检测细胞凋亡，以RT-PCR技术检测质粒转染前后生存素基因mRNA的表达。结果 pEGFP-C1-反义生存素重组质粒转染MKN-45低分化胃腺癌细胞株后细胞凋亡明显增加，G2/M期细胞减少，同时生存素基因在mRNA水平被抑制。结论 反义生存素核酸能抑制细胞增殖。减少生存素基因的mRNA表达而促进胃癌细胞凋亡。     

外源性FHIT基因表达对长春新碱诱导人胃癌MKN-28细胞凋亡的影响

目的:探讨外源性FHIT基因表达对长春新碱诱导的胃癌细胞凋亡的影响及其分子机制.方法:通过脂质体将重组FHIT基因PRC/CMV质粒和空载体转染到人胃癌细胞MKN-28,Western blot法检测外源性FHIT蛋白的表达;使用不同浓度的长春新碱分别处理各组细胞,MTT法分析细胞增殖,流式细胞术检测细胞的凋亡:Western blot法检测细胞Bcl-2和Bax的表达.结果:转染FHIT基因后,MKN-28细胞检测到FHIT蛋白的表达;长春新碱处理48 h后,转染FHIT基因组细胞、转染空载体组细胞及未转染组细胞的凋亡率分别是30.967%±2.122%、11.033%±1.724%、10.733%±1.021%,转染FHIT基因组细胞凋亡更明显(F=142.045,P<0.05);转染FHIT基因组细胞经长春新碱处理后Bcl-2蛋白表达减少,Bax蛋白表达增加.结论:外源性FHIT基因表达可以增强长春新碱诱导的胃癌细胞凋亡,其机制可能与凋亡相关蛋白Bcl-2和Bax表达有关.     

shRNA沉默干扰HMGA2基因对胃癌细胞株MKN-45的增殖与凋亡的影响

目的:观察小分子RNA(shRNA)沉默后HMG-A2基因在胃癌细胞株MKN-45的表达,并探讨HMGA2基因对胃癌细胞的增殖与凋亡的影响.方法:构建针对人HMGA2基因的shRNA真核表达载体,瞬时转染人胃癌细胞株MKN-45.用细胞免疫组化的方法观察转染72h后HMGA2的蛋白表达水平,以MTT比色法、流式细胞术检测转染后MKN-45细胞的生长增殖、凋亡的情况.结果:转染HMGA2-shRNA组的蛋白表达强度(171.34±19.61)明显弱于scrambled组(143.48±19.04)和空白对照组(141.79±18.09,P<0.05),较之scrambled组(5.66%±0.63%)和空白对照组,HMGA2-shRNA组(39.32%±2.37%)能明显抑制细胞的增殖(P<0.05),HMGA2-shRNA组的凋亡率(39.67%±2.35%)与scrambled组(4.29%±1.33%)和空白对照组(5.05%±1.84%)相比明显增加(P<0.05).结论:靶向HMGA2基因的shRNA可以有效抑制人胃癌MKN-45细胞的生长,并促进细胞的凋亡,HMGA2可能是胃癌治疗的一个潜在靶点.     

粘蛋白1介导的抗胃癌细胞MKN-45侵袭作用的研究

目的 构建粘蛋白1 (MUC1)基因的反义肽核酸(PNA),观察对胃癌细胞MKN-45侵袭能力的影响,并探讨其机制.方法 根据MUC1基因的序列设计反义PNA序列,经脂质体介导转染胃癌细胞MKN45,并设空载体组(随机对照)和空白对照组(阴性对照)运用荧光定量PCR检测MUC1的表达,并观察E-粘附素的表达变化;Transwell小室实验观察对胃癌细胞侵袭力的影响.结果 构建的3个MUC1基因的反义PNA均能有效抑制该基因表达,其表达水平分别为0.62±0.18,0.49±0.12和0.60±0.21,显著低于阴性对照组(1.18±0.03,P＜0.01).阴性对照组与随机对照组之间差异无统计学意义(1.00±0.04,P=0.657).取抑制效率最高的PNA进行后续研究转染MUC1 PNA后的胃癌细胞侵袭能力显著下降(t=2.09,P=0.005),并伴有E-粘附素基因及蛋白的表达上调.结论 胃癌细胞MKN-45中MUC1基因与E-粘附素的表达存在负相关,抑制MUC1基因的表达能显著抑制肿瘤细胞的侵袭能力.     

siRNA沉默Livin基因对胃癌细胞生长、凋亡的影响

目的:观察小分子干扰RNA(siRNA)沉默Livin基因在胃癌BGC-823细胞中的表达, 并探讨Livin基因对胃癌细胞生长、凋亡的影响.方法:自行设计两条针对Livin基因的siRNA:Livin-sh-1和Livin-sh-2, 以此构建相应的表达载体并分别转染至对数生长期胃癌BGC-823细胞, 经G418筛选后分别采用半定量RTPCR检测不同siRNA实验分组细胞BGC-823mRNA水平变化, 四氮唑盐比色法(MTT)检测细胞增殖、流式细胞仪检测胃癌细胞的凋亡.结果:siRNA对照组与空siRNA载体组Livinα/β mRNA表达差别无显著性; 但转染siRNA组Livin α/β mRNA表达显著低于空白对照组和空siRNA载体组(Livin α:0.11±0.07 vs 0.37±0.10, 0.34±0.08; Livin β:0.13±0.04 vs 0.43±0.09, 0.45±0.11, 均P<0.05). 空白对照组与空siRNA载体组相比, 24、48、96 h和1 wk时细胞生长未受影响; 而siRNA组在转染后24 h和48 h细胞生长未受影响, 但在96 h和1 wk时则被明显抑制( P<0.01). 转染siRNA组的细胞的凋亡率与空白对照组和转染空siRNA载体组相比显著增加(14.85%±1.35% vs 4.51%±0.36%, 6.13%±0.71%, 均P<0.05).结论:siRNA沉默Livin基因能抑制胃癌细胞的生长, 促进胃癌细胞的凋亡, Livin基因有可能成为胃癌治疗的新靶点.     

人宫颈癌基因反义核酸抑制肝癌细胞生长作用的实验研究

目的探讨针对人宫颈癌基因(HCCR)反义核苷酸影响肝癌细胞增殖、凋亡的作用。方法构建pcD-NA3.1-HCCR反义真核表达质粒,转染HepG2细胞,实时定量PCR检测HCCR mRNA表达水平,Western blot检测其蛋白表达水平,流式细胞仪观察HepG2细胞凋亡和细胞周期的变化,MTT法检测细胞的增殖活性。结果HCCR反义核酸可有效抑制HepG2细胞HCCR的表达,与转染前相比,转染后24小时HCCR mRNA水平下降到16%,其蛋白表达水平在24小时也明显降低。对细胞的增殖与凋亡活性检测显示:转染HCCR反义质粒后,HepG2细胞增殖受到抑制,转染后24小时抑制率为47.62%(P<0.05),细胞凋亡率为14.34%±0.91%,较对照组明显增多(t=21.799,P<0.05),细胞分裂多停止在G0G1期。结论HCCR反义核酸可以抑制HepG2细胞中HCCR的表达,抑制细胞增殖,促进细胞凋亡,使细胞停滞于G1期。HCCR反义核酸用于肝癌细胞的基因治疗具有一定的潜在意义。     

Smac基因过表达联合顺铂对SMMC-7721细胞增殖和凋亡的影响

目的 探讨Smac基因过表达联合顺铂对肝癌细胞增殖和凋亡的影响.方法 采用脂质体介导的转染方法 ,将重组质粒pcDNA3.1+hSmac导入人肝癌细胞株SMMC-7721中,采用Western blot和流式细胞术检测转染前后Smac蛋白表达情况.转染24 h后分别加入终浓度为5、15、25 μg/ml的顺铂诱导细胞凋亡.采用四甲基偶氮唑盐比色法检测癌细胞的增殖抑制作用,吖啶橙-溴化乙锭荧光染色法和膜联蛋白V/碘化丙啶双染标记流式细胞术检测细胞凋亡. 结果 Westernblot和流式细胞术检测结果证实转染后Smac蛋白表达明显增加(P＜0.01).与空白对照相比,Smac基因过表达可抑制癌细胞增殖,促进凋亡(P＜0.01).而且给予顺铂处理后,与空白对照组相比,细胞生长抑制率随剂量增加而显著上升,且转染Smac的细胞生长抑制率较相应未转染Smac的细胞明显升高(P＜0.01).吖啶橙-溴化乙锭荧光染色法和流式细胞术检测显示,转染Smac加顺铂处理组较单纯顺铂处理组细胞凋亡明显增加,差异具有统计学意义(P＜0.01).这表明Smac基因过表达可增强顺铂对肝癌细胞的增殖抑制和凋亡促进作用. 结论 促凋亡基因Smac可在肝癌细胞中过表达,抑制癌细胞的增殖和促进凋亡;而且过表达的Smac基因可增强癌细胞对化疗药物顺铂的敏感性,这为研究Smac在癌细胞凋亡过程中的调控作用以及肝癌化学治疗效果的改善提供了实验基础.     

脂质体介导的VEGF-C反义寡核苷酸对肺腺癌细胞系 A549生长、凋亡的影响

目的 探讨脂质体介导的血管内皮生长因子C(VEGF-C) 反义寡核苷酸对肺腺癌细胞系 A549生长、凋亡的作用.方法 将 A549 细胞随机分为磷酸盐缓冲液对照组(PBS组)、正义寡核苷酸对照组(SODN组)和反义寡核苷酸转染组(AODN组),应用细胞计数方法计数每组细胞数,苏木精-伊红(HE)染色法观察细胞形态及分布,流式细胞仪分析A549细胞凋亡情况.应用Western印迹方法检测转染后各组A549细胞中VEGF-C蛋白表达.结果 VEGF-C反义寡核苷酸转染后,AODN组细胞生长受抑程度显著高于对照组,差异有统计学意义(P<0.01),生长缓慢且核分裂像较对照组明显减少.流式细胞仪检测结果显示AODN组细胞凋亡率明显高于对照组,差异有统计学意义(P<0.01).Western印迹结果显示VEGF-C反义寡核苷酸可明显抑制VEGF-C基因表达.结论 脂质体介导的VEGF-C反义寡核苷酸能有效地干扰VEGF-C基因表达,并可抑制肺腺癌A549细胞生长并促进其凋亡.     

Inhibition of human telomerase in MKN—45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest

AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.     

靶向ING1基因的miR-622真核表达载体在胃癌细胞MKN-45中的鉴定及其功能

目的:研究构建靶向ING1基因的miR-622真核表达载体并验证其转染人胃癌细胞株MKN-45细胞后对ING1基因的干扰效果及其功能.方法:将外源性重组真核表达载体pSuper/miR-622转染到人胃癌细胞株MKN-45内,经G418筛选并建立高表达miR-622的稳定转染胃癌细胞株.稳定表达该miR-622的胃癌细...     

Stable transfection of extrinsic Smac gene enhances apoptosis-inducing effects of chemotherapeutic drugs on gastric cancer cells

AIM: To explore the feasibility of enhancing apoptosis-inducing effects of chemotherapeutic drugs on human gastric cancer cells by stable transfection of extrinsic Smac gene. METHODS: After Smac gene was transferred into gastric cancer cell line MKN-45, subclone cells were obtained by persistent G_(418) selection. Cellular Smac gene expression was determined by RT-PCR and Western blotting. After treatment with mitomycin (MMC) as an apoptotic inducer, in vitro cell growth activities were investigated by trypan blue-staining method and MTT colorimetry. Cell apoptosis and its rates were determined by electronic microscopy, annexin V-FTTC and propidium iodide staining flow cytometry. Cellular caspase-3 protein expression and its activities were assayed by Western blotting and colorimetry. RESULTS: When compared with MKN-45 cells, the selected subclone cell line MKN-45/Smac had significantly higher Smac mRNA (3.12±0.21 vs 0.82±0.14, t=7.52, P＜0.01) and protein levels (4.02±0.24 vs0.98±0.11, t=8.32, P＜0.01). After treatment with 10 μg/mL MMC for 6-24 h, growth inhibition rate of MKN-45/Smac (15.8±1.2-54.8±2.9%) was significantly higher than that of MKN-45 (5.8±0.4-24.0±1.5%, t=6.42, P＜0.01). Partial MKN-45/Smac cancer cells presented characteristic morphological changes of apoptosis under the electronic microscope with an apoptosis rate of 36.4±2.1%, which was significantly higher than that of MKN-45 (15.2±0.8%, t=9.25, P＜0.01). Compared with MKN-45, caspase-3 expression levels in MKN-45/Smac were improved significantly (3.39±0.42 vs0.96±0.14, t=8.63, P＜0.01), while its activities were 3.25 times as many as those of MKN-45 (0.364±0.010 vs0.112±0.007, t=6.34, P＜0.01). CONCLUSION: Stable transfection of extrinsic Smac gene and its over-expression in gastric cancer cell line can significantly enhance cellular caspase-3 expression and activities, ameliorate apoptosis-inducing effects of mitomycin C on cancer cells, which is a novel strategy to improve chemotherapeutic effects on gastric cancer.     

hnRNPA1在人胃癌细胞株BGC-823中的生物学功能

核不均一核糖核蛋白A1（hnRNPAl）是体内重要的RNA结合蛋白,通过调节pre-mRNA和mRNA参与转录和转录后调控过程,与肿瘤发生、发展密切相关。目前对hnRNPAl与胃癌的关系尚不十分清楚。目的：探讨hnRNPAl在人胃癌细胞中的生物学功能,明确其与胃癌的关系。方法：以蛋白质印迹法检测正常人胃黏膜上皮细胞株GES一1和人胃癌细胞株MKN-28、SGC-7901、BGC-823中的hnRNPAl表达。构建靶向hnRNPAl基因的siRNA重组质粒并稳定转染BGC一823细胞,以稳转空质粒pU6的细胞株作为对照,蛋白质印迹法验证干扰效果。分别以CCK一8实验、细胞划痕实验、Transwell侵袭实验和流式细胞术分析各组BGC-823细胞的增殖、迁移、侵袭能力和细胞凋亡情况。结果：hnRNPA1在3株人胃癌细胞株中均呈高表达,BGC-823细胞表达水平最高。与pU6稳转组相比,siRNAhnRNPAl稳转组BGC．823细胞增殖抑制率为36．3％,细胞迁移[（5．44±0．25）斗In／h对（9．37±0．49）μm／h,P〈0．01]和侵袭（穿膜细胞数146．30±12．56对312．51±9．62,P〈0．01）受抑,细胞凋亡率降低（3．6％±0．3％对12．7％±0．2％,P〈0．01）。结论：hnRNPAl在人胃癌细胞中呈高表达,其可能通过促进肿瘤细胞增殖、迁移、侵袭,在胃癌侵袭和转移过程中发挥作用。     

靶向XIAP的shRNA重组质粒的构建

目的:设计以X染色体连锁凋亡抑制蛋白(XIAP)为靶向的shRNA,构建携带此shRNA的重组质粒,检测其抑制XIAP表达的效应,筛选RNA干扰作用最强的shRNA片段.方法:设计4对针对XIAP基因不同位点的shRNA片段,构建携带此shRNA片段的真核表达载体psiRNA-Hhneo-XIAP,通过脂质体介导的方法将重组质粒转染到肝癌细胞株HepG2细胞中.采用逆转录酶链式反应(RT-PCR)及蛋白印迹(Western blotting)方法检测XIAP的mRNA及蛋白表达情况,比较转染前后其表达差异,以判断各shRNA的干扰效应.结果:成功构建含shRNA片段的重组质粒.经质粒测序证实,插入的DNA片段的序列与设计序列完全一致.重组质粒转染HepG2细胞后,XIAP基因的mRNA水平及蛋白表达水平明显下调,其中以1号重组质粒效应最强.shRNA作用48h后,对HepG2细胞中XIAP mRNA和蛋白的抑制率与3,4号重组质粒相比,均具有显著性差异(mRNA:94.5% vs 81.5%,82.6%,均P<0.01:蛋白:92.6% vs 80.7%,82.9%,均P<0.01).结论:成功构建了携带以XIAP为靶向的shRNA的重组质粒,其对肝癌细胞内XIAP的表达具有显著抑制效应.X染色体连锁凋亡抑制蛋白;;短发夹状RNA;;肝癌;;逆转录聚合酶链式反应;;免疫蛋白印迹     

Synergistic effect of oxymatrine and angiogenesis inhibitor NM-3 on modulating apoptosis in human gastric cancer cells

AIM: To investigate the synergistic effect of oxymatrine(OM) and angiogenesis inhibitor NM-3 on modulatingapoptosis in human gastric cancer cell lines SGC-7901,MKN-45, MKN-74. METHODS: Human gastric cancer lines SGC-7901,MKN-45, MKN-74 were treated with OM in the absenceand presence of NM-3. The inhibitory rates weredetected by MTT assay. Synergistic effect of OM andNM-3 on the growth of survivin, bcl-2, bax and p53 inSGC-7901 cells were examined by semiquantitative RT-PCR and Western blotting, and their growth inhibitoryeffects were also observed on SGC-7901 tumor xenograftin nude mice.RESULTS: OM combined with NM-3 exhibited asynergistic inhibitory effect on the growth of SGC-7901,MKN-45 and MKN-74 cells in a time-dependent manner.Twenty-four hours after treatment with OM, NM-3 aloneand their combination, mRNA expression of survivin andbcl-2 in SGC-7901 cells decreased, p53 mRNA expressionincreased. OM (4 g/L) combined with NM-3 significantlyincreased the expression of p53 mRNA and decreasedthe expression of survivin and bcl-2 compared witheither agent alone (193% ± 34% vs 129% ± 12%;44% ± 18% vs 92% ± 18%; 36 ± 17% vs 93% ± 23%,P < 0.05). Western blotting showed that the synergisticeffect of OM and NM-3 on protein translation of survivin,bcl-2 and p 53 was in accordance with their mRNAs.Furthermore, OM/NM-3 combination obviously exhibitedantitumor growth effect in xenografted human gastriccancer cells SGC-7901 compared with either agent alone.CONCLUSION: OM combined with NM-3 has synergisticinhibitory effects on human gastric cancer cells in vitro and can suppress the growth of xenografted human gastric cancer cells SGC-7901 in vivo.     

Lobaplatin inhibits growth of gastric cancer cells by inducing apoptosis

AIM: To assess the anti-cancer effect of lobaplatin on human gastric cancer cells, and to explore the underlying molecular mechanisms.METHODS: The human gastric cancer cell lines MKN-28, AGS and MKN-45 were used. The cytotoxicity of lobaplatin was detected using an MTS cell proliferation assay. Flow cytometry was used to detect cell apoptosis using Annexin V-FITC Apoptosis Detection Kit. The expression of apoptosis-regulated genes was examined at the protein level using Western blot.RESULTS: Lobaplatin inhibited the proliferation of human gastric cancer cells and induced apoptosis, which may be associated with the up-regulation of Bax expression, poly(ADP-ribose) polymerase (PARP) cleavage, p53 expression and the reduction of Bcl-2 expression.CONCLUSION: The cytotoxicity of lobaplatin may be due to its ability of inducing apoptosis of gastric cancer cells, which would support the potential use of lobaplatin for the therapy of gastric cancer.     

缬草波春诱导MKN-45胃癌细胞凋亡

目的:研究缬草波春诱导胃癌细胞凋亡,探讨其诱导凋亡与半胱氨酸酶(Caspase)及生存素(Survivin)mRNA、P53蛋白、Survivin蛋白表达的关系.方法:以100 mg/L的缬草波春作用于加Caspase-3抑制剂、Caspase-8抑制剂、Caspase-9抑制剂和未加Caspases抑制剂培养的MKN-45细胞24,48和72 h,用流式细胞仪分别检测细胞凋亡率;不同浓度的缬草波春(5,10,25,50,100 mg/L)作用MKN-45细胞不同时间(24,48,72 h)后,用tripure提取液提取细胞RNA,用RT-PCR法,检测Survivin mRNA的表达.不同浓度缬草波春(50和100 mg/L)作用MKN-45胃癌细胞株24 h后,用免疫组化的方法,检测P53蛋白和Survivin蛋白的表达.结果:单用Caspase抑制剂组,作用24,48和72 h对MKN-45细胞凋亡率无明显影响,与对照组比较差异无显著意义.Caspase-3抑制剂、Caspase-9抑制剂与缬草波春联合应用后24,48和72 h使MKN-45细胞凋亡率高于对照组(24 h:5.73%,5.41% vs 4.38%,P＜0.01;48 h:6.88%,6.32% vs 4.35%,P＜0.01;72 h:7.72%,8.62% vs 4.54%,P＜0.01),低于缬草波春组(24 h:5.73%,5.41% vs 8.14%,P＜0.01;48 h:6.88%,6.32% vs 12.31%,P＜0.01;72 h:7.72%,8.62% vs 26.41%,P＜0.01),与对照组及缬草波春组比较差异均有显著意义(P＜0.01).Caspase-8抑制剂与缬草波春联合应用后24,48和72 h MKN-45细胞凋亡率明显增加,与对照组比较差异有显著意义(8.02% vs 4.38%,P＜0.01;11.05% vs 4.35%,P＜0.01;24.86% vs 4.54%,P＜0.01),与单用缬草波春组比较差异无显著意义.缬草波春降低MKN-45胃癌细胞株Survivin mRNA的表达,并有浓度依赖性和时间依赖性,而且使MKN-45胃癌细胞株P53蛋白表达增加,Survivin蛋白表达降低,均有浓度依赖性.结论:缬草波春可诱导MKN-45细胞凋亡,其作用可部分被Caspase-3,Caspase-9抑制剂所抑制,但不能被Caspase-8抑制剂所抑制.缬草波春诱导MKN-45胃癌细胞株凋亡与P53蛋白表达提高及Survivin mRNA和Survivin蛋白低表达降低有关.     

Inhibitory effect of human telomerase antisense oligodeoxyribonucleotides on the growth of gastric cancer cell lines in variant tumor pathological subtype

AIM: To investigate the inhibitory effect of specialized human telomerase antisense oligodeoxyribonucleotides on the growth of well (MKN-28), moderately (SGC-7901) and poorly (MKN-45) differentiated gastric cancer cell lines under specific conditions and its inhibition mechanism, and to observe the correlation between the growth inhibition ratio and the tumor pathologic subtype of gastric cancer cells. METHODS: Telomerase activity in three gastric cancer cell lines of variant tumor pathologic subtype was determined by modified TRAP assay before and after the specialized human telomerase antisense oligodeoxyribonucleotides were dealt with under specific conditions. Effect of antisense oligomer under specific conditions of the growth and viability of gastric cancer cell lines was explored by using trypan blue dye exclusion assay, and cell apoptosis was detected by cell morphology observation, flow cytometry and TUNEL assay. RESULTS: Telomerase activity was detected in well, moderately and poorly differentiated gastric cancer cell lines (the quantification expression of telomerase activity was 43.7TPG, 56.5TPG, 76.7TPG, respectively). Telomerase activity was controlled to 30.2TPG, 36.3TPG and 35.2TPG for MKN-28, SGC-7901 and MKN-45 cell lines respectively after treatment with human telomerase antisense oligomers at the concentration of 5 μmol/L, and was entirely inhibited at 10 μmol/L, against the template region of telomerase RNA component, whereas no inhibition effect was detected in missense oligomers (P<0.05). After treatment with antisense oligomers at different concentrations under specific conditions for 96 h, significant growth inhibition effects were found in MKN-45 and SGC-7901 gastric cancer cell lines (the inhibition ratio was 40.89% and 71.28%), but not in MKN-28 cell lines (15.86%). The ratio of inactive SGC-7901 cells increased according to the prolongation of treatment from 48 to 96 h. Missense oligomers could not lead to the same effect (P<0.05). Apoptosis of SGC-7901 and MKN-45 cells was detected not only by morphology and TUNEL assay but also by flow cytometry. The apoptotic rate reached 33.56% for SGC-7901 cells and 44.75% for MKN-45 cells. CONCLUSION: The viability and proliferation of gastric cancer cells can be inhibited by antisense telomerase oligomers. The growth inhibition of gastric cancer cells is correlated with concentration, time and sequence specialty of antisense oligomers. The inhibition mechanism of antisense human telomerase oligomers depends not only on the sequence specialty but also on the biological characteristics of gastric cancer cell lines.