反义人类端粒酶RNA逆转录病毒载体构建及其对结直肠癌细胞的抑制作用

Construction of antisense human telomeric RNA retroviral vector and its inhibition on colorectal cancer cells

目的研究反义人类端粒酶RNA逆转录病毒载体对结直肠癌HT29细胞端粒酶活性及细胞生长的抑制作用,探讨以端粒酶为靶点的结直肠癌基因治疗的可能性。方法将端粒酶hTR的cDNA反向插入逆转录病毒载体pLXRN中,转染包装细胞PT67后获得反义重组病毒,感染结直肠癌HT29细胞。采用RT-PCR检测hTR表达,端粒酶重复扩增法(telomeraserepeatamplificationprotocol,TRAP)检测端粒酶活性,绘制生长曲线了解细胞生长状态,倒置显微镜观察和DNA片段电泳检测细胞凋亡。结果反义hTR作用后的结直肠癌细胞hTR表达下降,端粒酶活性和细胞生长受到明显抑制,细胞出现凋亡。结论反义hTR对结直肠癌HT29细胞的生长和端粒酶活性具有明显的抑制作用,有可能以hTR为靶点对结直肠癌进行基因治疗。

Objective To investigate the inhibition of telomerase activity and cellular proliferation in colorectal cancer HT29 cell lines by antisense human telomeric RNA(hTR) treatment, and explore the possibility of the telomerase as a target of gene therapy for colorectal cancer. Methods The replication deficient retrovirus expressing telomeric antisense RNA was constructed to infect the HT29 cells. hTR expression was examined by RT PCR. Telomerase activity was assayed by telomerase repeat amplification protocol (TRAP). Cell proliferation was investigated by cellular growth curve. Cellular apoptosis was detected by DNA electrophoresis method and invert microscope. Results hTR expression and telomerase activity of colorectal cancer HT29 cells were significantly inhibited, cellular proliferation were arrested, and cellular apoptosis occurred after antisense hTR transduction. Conclusions The transduction of antisense hTR by retroviral vector can significantly inhibit the hTR expression and telomerase activity of colorectal cancer HT29 cell lines, arrest the cellular proliferation and promote cellular apoptosis. The hTR may be a potential target of gene therapy for colorectal carcinoma.