CAJ | 학술논문

目的探讨慢病毒介导的端粒酶逆转录酶(hTERT)小干扰RNA(siRNA)对肝癌细胞生长的影响.方法构建携带hTERT siRNA的慢病毒表达载体,在体外转染人肝癌HepG2细胞系,以MTT法测定细胞生长变化,半定量逆转录聚合酶链反应(RT-PCR)检测hTERT mRNA的表达.将转染hTERT siRNA的HepG2细胞,接种于裸鼠腋窝皮下,观察移植瘤生长情况.取移植瘤组织,常规HE染色,行组织学观察,应用原位末端标记(TUNEL)技术检测细胞凋亡情况.结果 hTERT siRNA转染HepG2细胞后,随着时间的延长,对细胞增殖的抑制作用逐渐增强,到第8天,干扰组细胞抑制率为57.5%,与对照组比较,差异有统计学意义(P<0.01).RT-PCR产物凝胶电泳图显示,hTERT siRNA转染后,肿瘤细胞的端粒酶活性明显下降.转染后72 h,干扰组hTERT mRNA的表达水平为0.035±0.007,明显低于空载体对照组(0.151±0.016)和空白对照组(0.155±0.014,均P<0.01).转染细胞皮下接种后,干扰组的肿瘤生长速度明显慢于两个对照组,随着时间的延长,差异越来越明显.移植瘤组织常规HE染色显示,组织坏死增多,周围炎性细胞浸润.TUNEL检测结果显示,细胞凋亡增多,增殖减慢.结论慢病毒介导的hTERT siRNA转染能明显抑制肝癌细胞的生长.
목적 탐토만병독개도적단립매역전록매(hTERT)소간우RNA(siRNA)대간암세포생장적영향.방법 구건휴대hTERT siRNA적만병독표체재체,재체외전염인간암HepG2세포계,이MTT법측정세포생장변화,반정량역전록취합매련반응(RT-PCR)검측hTERT mRNA적표체.장전염hTERT siRNA적HepG2세포,접충우라서액와피하,관찰이식류생장정황.취이식류조직,상규HE염색,행조직학관찰,응용원위말단표기(TUNEL)기술검측세포조망정황.결과 hTERT siRNA전염HepG2세포후,수착시간적연장,대세포증식적억제작용축점증강,도제8천,간우조세포억제솔위57.5%,여대조조비교,차이유통계학의의(P<0.01).RT-PCR산물응효전영도현시,hTERT siRNA전염후,종류세포적단립매활성명현하강.전염후72 h,간우조hTERT mRNA적표체수평위0.035±0.007,명현저우공재체대조조(0.151±0.016)화공백대조조(0.155±0.014,균P<0.01).전염세포피하접충후,간우조적종류생장속도명현만우량개대조조,수착시간적연장,차이월래월명현.이식류조직상규HE염색현시,조직배사증다,주위염성세포침윤.TUNEL검측결과현시,세포조망증다,증식감만.결론 만병독개도적hTERT siRNA전염능명현억제간암세포적생장.
Objective To study the efficacy of anti-telomerase siRNA in hepatocellular carcinoma both in vitro and in vivo. Methods Lentvims vectors contained anti-telomerase siRNA were conducted with a high performance homologous recombination system, and then were transduced into human hepatocellular carcinoma HepG2 cells. The telomerase activity was detected by RT-PCR, HepG2 cell proliferation was determined by MTT assay, and apoptosis was detected by TUNEL assay. The in vivo experiment was carried out by inoculation of HepG2 cells into nude mice and the tumor growth was measured and analyzed. Results The growth of transfected HepG2 cells was significantly inhibited and the inhibition rate was 57.5% at the 8th day after transfection. The telomerase activity was significantly suppressed in vitro. The growth of transfected human hepatocellular HepG2 tumor in the nude mice was also significantly inhibited. Conclusion The results of this study demonstrate that the growth of hepatocellular carcinoma cells is effectively inhibited by transfection of anti-telomerase siRNA both in vitro and in vivo.