Survvin反义寡核苷酸诱导人胃癌细胞凋亡的作用

目的探讨生存素（Survivin）反义寡核苷酸（ASODN）诱导人胃癌细胞SGC7901凋亡的作用。方法设计合成特异性靶向Survivin的ASODN,将胃癌细胞株分为空白对照组（Sham组）、脂质体转染对照组（Lip组）、正义链转染对照组（Lip-SODN组）和ASODN转染组（Lip-ASODN组）。作用48h后,Westemblot法检测各组Survivin表达情况,流式细胞仪检测各组细胞凋亡率,免疫组化SP法检测细胞中PCNA表达情况。结果脂质体介导Survivin ASODN转染后的胃癌细胞Survivin蛋白表达明显下降;ASODN转染组细胞凋亡率明显高于各对照组（P均〈0．05）,各对照组间无统计学差异（P〉0．05）。ASODN转染后胃癌细胞中PCNA表达水平明显降低。结论 Survivin ASODN转染胃癌细胞能下调Survivin蛋白表达,诱导胃癌细胞凋亡,抑制细胞增殖,具有明显的抗癌作用。     

端粒酶反义寡核苷酸促进顺铂诱导原代胃癌细胞凋亡

目的:探讨端粒酶反义寡核苷酸(PS-ASODN)对顺铂诱导原代胃癌细胞凋亡的影响。方法:采用台盼蓝拒染法计算细胞生长抑制率,观察PS-ASODN联合顺铂对原代胃癌细胞生长的影响;流式细胞学观察细胞凋亡率及细胞周期变化。结果:终浓度为3μM的PS-ASODN作用于原代胃癌细胞24 h后加入顺铂(2.0μg/ml),能明显抑制胃癌细胞增殖,同时流式细胞学检测到凋亡峰,细胞受阻于G0/G1期,作用48 h及72 h的凋亡细胞百分率(35.1％,45.7％)明显高于N-ASODN组、PS-ASODN组、DDP组及N-ASODN+DDP组,差异有显著性(P<0.05)。其作用呈时间依赖性及序列特异性。结论:以端粒酶RNA模板区为靶点的反义寡核苷酸可促进顺铂诱导的胃癌细胞凋亡,对胃癌具有重要治疗价值。     

survivin反义寡核苷酸转染肝癌细胞可诱导caspase-3活化

目的:观察survivin反义寡核苷酸(antisense oligonucleotide,AS ODN)诱导肝癌细胞凋亡的可能途径。方法:培养survivin表达阳性的肝癌细胞株SMMC-7721。设计合成特异性靶向survivin的反义寡核苷酸。将SMMC-7721细胞接种于6孔培养板内,分为6组:①空白对照组;②脂质体转染对照组;③正义链转染对照组;④200nmol/L AS ODN转染组;⑤400nmol/L AS ODN转染组;⑥600nmol/L AS ODN转染组。作用20小时后收获各组细胞,并进行后续实验:①倒置显微镜下观察细胞形态变化;②western blot法检测各组细胞survivin蛋白的表达情况;③流式细胞术检测细胞增殖和凋亡指数;④比色法检测各组细胞caspase-3的活性变化。结果:①AS ODN转染组细胞survivin表达减弱,以600nmol/L转染组最为明显,各对照组survivin蛋白表达无明显改变;②AS ODN转染组细胞形态出现变圆、折光增强、漂浮、细胞碎片形成等变化,而各对照组细胞生长良好;③各AS ODN转染组细胞凋亡指数明显高于各对照组(P<0.05),以600nmol/L转染组最为明显,而各对照组间差异无显著性意义(P>0.05);④各AS ODN转染组细胞增殖指数明显低于各对照组(P<0.05),以600nmol/L转染组最为明显,而各对照组间差异无显著性意义;⑤在各AS ODN转染组中,可见到不同程度的caspase-3的活化,而对照组细胞内无明显的变化。结论:survivin AS ODN转染能下调survivin蛋白表达,诱导肝癌细胞凋亡,抑制细胞增殖,其作用途径可能涉及抑制survivin表达,诱导caspase-3活化,启动凋亡信号传导。     

脂质体介导的反义寡核苷酸抑制人胃癌细胞端粒酶活性

目的：探讨特异性反义寡脱氧核苷酸（ａｎｔｉｓｅｎｓｅ ｏｌｉｇｏｄｅｏｘｙｎｕｃｌｅｏｔｉｄｅ,ＡＳＯＤＮ）对人胃癌细胞端粒酶活性的影响。方法应用脂质体包裹与端粒酶ＲＮＡ模板区互补的ＡＳＯＤＮ,处理人胃癌细胞系ＦＧＣ８５。用端粒重复扩增分析（ｔｅｌｏｍｅｒｅ ｒｅｐｅａｔ ａｍｐｌｉｆｉｃａｔｉｏｎ ｐｒｏｔｏｃｏｌ,ＴＲＡＰ）－ＰＣＲ－ＥＬＩＳＡ检测法观察端粒酶活性的变化,结果经脂质体包裹ＡＳＯ     

反义寡核苷酸转染对SGC-7901胃癌细胞端粒酶活性及细胞凋亡的影响

目的：探讨端粒酶反义寡核脱氧核苷酸（PS－ASODN）对SGC－7901胃癌细胞端粒酶活性及细胞凋亡的影响。方法：脂质体介导的PS－ASODN作用于SGC－7901细胞后，台盼蓝拒染法计算细胞生长抑制剂，采用半定量TRAP－银染法检测端粒酶活性，用流式细胞仪观察细胞凋亡率及细胞周期变化，采用光镜及电镜观察细胞凋亡形态。结果：终浓度为3、2及1μM的PS－ASODN对SGC－7901细胞均有抑制作用，抑制率与对照组比较，差异有显著性（P＜0．01）；PS－ASODN转染后48h，端粒酶活性明显降低（P＜0．05）。以上抑制作用呈剂量依赖性及序列特异性。流式细胞仪检测到凋亡峰，细胞受阻于G0/G1期；光镜及电镜显示典型的细胞凋亡形态改变。对照寡核苷元上述作用。结论：以端粒酶RNA模板区为靶点的反义寡核苷不但明显抑制胃癌细胞的增殖，降低端粒酶活性，而且具有促凋亡作用，对胃癌具有重要治疗价值。     

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。     

Survivin反义寡核苷酸诱导肝癌细胞凋亡的作用

目的 用反义寡核苷酸封闭肝癌细胞中survivin基因的表达,研究其诱导细胞凋亡的作用及其机制。方法 用脂质体介导survivin反义寡核苷酸转染人肝癌细胞株SMMC-7721细胞,流式细胞仪检测细胞周期变化及细胞凋亡比率,激光共聚焦显微镜观察细胞骨架微丝系统变化,激酶活性检测方法测定细胞内caspase-3活性变化,免疫沉淀法测定细胞内丝裂原活化蛋白激酶p38(p38MAPK)活性的变化。结果 脂质体介导survivin反义寡核苷酸转染肝癌细胞后Ⅰ～Ⅵ组(空白对照、正义对照、400、600、800、1000ng/ml反义转染组)细胞内p38MAPK活性分别为7.03、7.07、13.47、16.37、43.97及47.87;caspase-3活性分别为0.015±0.010、0.014±0.002、0.026±0.003、0.042±0.001、0.093±0.001及0.100±0.001;Ⅳ～Ⅵ组细胞内p38MAPK及caspase-3活性较对照组明显升高。转染后细胞发生G_2/M期阻滞,Ⅰ～Ⅵ组细胞凋亡率分别为0.70％、0.76％、2.43％、7.82％、23.11％及31.35％,各实验组细胞凋亡较对照组明显增加。细胞内微丝形态结构破坏,Ⅰ～Ⅵ组细胞肌动蛋白平均荧光强度分别为189.69±6.68、184.23±8.76、173.14±8.15、99.48±6.57、76.69±10.05及63.80±6.79,Ⅳ～Ⅵ组细胞肌动蛋白平均荧光强度较对照组明显降低。结论 脂质体介导转染survivi     

蟾蜍毒素对人胃癌细胞凋亡的诱导机制

目的:研究蟾蜍毒素诱导人胃癌细胞MGC803凋亡及对凋亡因子Livin、caspase-3的影响.方法:应用MTT法检测10、20、40、80、160nmol/L蟾蜍毒素对胃癌细胞MGC803增殖抑制作用:瑞氏-吉姆萨染色法观察细胞形态学的变化;流式细胞术分析周期阻滞和细胞凋亡;Western blot法检测Livin...     

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.     

脂质体包裹EGFR反义寡脱氧核苷酸抑制人大肠癌细胞的增殖

目的研究用表皮生长因子受体（ＥＧＦＲ）反义寡脱氧核苷酸（ＡＳＯＤＮ）抑制人大肠癌细胞株ＨＲ８３４８的生长增殖，探讨大肠癌治疗的新途径．方法人工合成的ＥＧＦＲ反义ＯＤＮ及无关对照ＯＤＮ经脂质体包裹后作用ＨＲ８３４８细胞，采用ＭＴＴ法，［３Ｈ］ＴｄＲ掺入试验，细胞周期分析，裸鼠体内接种等方法测定细胞体内外增殖的变化．结果经ＥＧＦＲ反义ＯＤＮ处理的ＨＲ８３４８细胞与对照组ＨＲ８３４８细胞相比，体外增殖速度减慢（细胞增殖抑制率达７１％）、ＤＮＡ合成受抑（细胞增殖指数为０３６１ｖｓ０７８４，Ｓ期细胞数为３４８％ｖｓ５９９％）、裸鼠体内成瘤率下降（２５％ｖｓ１００％）．结论ＥＧＦＲ反义ＯＤＮ可有效抑制大肠癌细胞的体内外生长增殖，可用于实验性肿瘤基因治疗研究     

人脐带血间充质干细胞对人胃癌MGC80-3细胞增殖的影响

目的探讨人脐带血间充质干细胞(h UCB-MSCs)对人胃癌MGC80-3细胞体外增殖的影响。方法原代培养h UCBMSCs,通过流式细胞仪进行细胞表型鉴定;采用MTT法测定不同浓度h UCB-MSCs条件培养基作用于胃癌MGC80-3细胞后,胃癌细胞24 h、48 h、72 h的抑制率;倒置相差显微镜观察胃癌MGC80-3细胞形态的变化;流式细胞技术测定胃癌MGC80-3细胞凋亡率。结果采用流式细胞仪表型分析获得的第4代h UCB-MSCs均质性好;MTT法显示,不同浓度h UCB-MSCs条件培养基处理MGC80-3细胞24 h、48 h、72 h后,各实验组抑制率均有不同程度升高;倒置相差显微镜观察显示细胞形态发生变化;流式细胞术分析结果显示,MSC-CM能诱导MGC80-3细胞发生凋亡,细胞凋亡率随着MSC-CM的升高而升高,各实验组与空白对照组比较及各实验组之间比较,差异均有统计学意义(P0.05)。结论 h UCB-MSCs能明显抑制人胃癌细胞株MGC80-3细胞的体外增殖与转移,且作用效果具有剂量和时间依赖性。     

保护性自噬对5-FU诱导的胃癌细胞凋亡的抑制作用

目的:明确5-FU处理胃癌MGC803细胞后自噬的存在,并探讨自噬在5-FU诱导凋亡中的作用.方法:5-FU处理胃癌MGC803细胞.MTT检测细胞增殖能力.流式细胞术检测细胞凋亡.Western blot检测蛋白表达.荧光显微镜观察自噬的发生.结果:0.1-1000mg/L的5-FU作用MGC803细胞48h,抑制细...     

Inhibitory effect of a liposome‐mediated antisense oligodeoxynucleotide on telomerase activity in a human gastric cancer cell line

OBJECTIVE : To investigate the effect of a specific antisense oligodeoxynucleotide (ASODN) on the telomerase activity of the human gastric cancer cell line FGC₈₅. METHODS : FGC₈₅ cells were treated in vitro with ASODN complementary to the template region of the RNA component of human telomerase. A cationic liposomal transfection reagent was used as a delivery vector. Telomerase activity was detected by using the telomeric repeat amplification protocol/polymerase chain reaction/enzyme‐linked immunosorbent assay (TRAP‐PCR‐ELISA) method. RESULTS : The telomerase activity of FGC₈₅ cells was suppressed significantly by the liposome/ASODN treatment and the effect was related to the con‐centration of liposome/ASODN and treatment time. CONCLUSIONS : Specific ASODN have an inhibitory effect on telomerase activity in human gastric cancer cell line FGC₈₅. Specific ASODN could be used in research into gene therapy for cancer treatments and the exploration of carcinogenesis.     

下调CD44基因表达对人胃癌SGC7901细胞裸鼠移植瘤的抑制作用及其机制

目的探讨靶向CD44的短发夹RNA(shRNA)对人胃癌裸鼠移植瘤生长抑制作用及其机制。方法利用CD44 shRNA细胞及对照细胞株建立裸鼠原位移植瘤及皮下种植瘤模型,监测肿瘤生长变化,采用RT-PCR及免疫组织化学方法检测瘤体内CD44表达的变化,并进一步检测上皮-间质转化(EMT)相关因子表达的变化。结果成功构建了CD44 shRNA转染荷瘤皮下及原位裸鼠模型。与对照shRNA组、空白对照组分别比较,CD44 shRNA组荷瘤裸鼠肿瘤生长速度减慢,皮下种植瘤及原位移植瘤质量减轻,差异均有统计学意义(P均0.01)。RT-PCR结果发现,CD44 shRNA组CD44mRNA表达在皮下种植瘤及原位移植瘤均显著下调,差异均有统计学意义(P均0.01)。CD44 shRNA组细胞EMT相关基因E-cadherin表达增加,N-cadherin、Vimentin表达降低。结论 CD44 shRNA可以有效抑制人胃癌SGC7901细胞裸鼠移植瘤生长,并下调CD44的表达水平,这可能与CD44基因参与EMT相关。