慢病毒载体介导端粒酶逆转录酶小干扰RNA治疗肝癌的实验研究

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.

Inhibitory effect of lentivirus transduced anti-telomerase siRNA therapy on hepatocellular carcinoma in nude mice

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.