端粒酶小干扰RNA技术体外抑制Hut78细胞生长及诱导凋亡的研究

Effect of small interfering RNA directed against telomerase on the proliferation and apoptosis of Hut78 cells

目的探讨针对端粒酶逆转录酶(hTERT)基因的小干扰RNA(siRNA)技术对皮肤T细胞淋巴瘤(CTCL)细胞株Hut78端粒酶活性和细胞生长的影响。方法用T7 RNA聚合酶介导的体外转录法合成两对hTERT-siRNA,将合成的siRNA转入Hut78细胞中,用端粒酶重复序列扩增及酶联免疫吸附方法(TRAP-PCR-ELISA)和逆转录聚合酶链反应(RT-PCR)分别检测Hut78细胞端粒酶活性及hTERT mRNA的表达。用MTT法和流式细胞仪分析细胞增殖及凋亡的变化。结果将合成的siRNA(250 ng)转染入Hut78细胞48 h后,端粒酶活性被明显抑制,抑制率分别为68%和64%;hTERT mRNA的表达下降。细胞生长抑制作用有明显的剂量依赖性,以1000 ng剂量抑制效果最明显。同时观察到细胞凋亡,凋亡率分别为15.86%和11.10%(P>0.05)。结论体外转录法合成的hTERT-siRNA能明显抑制Hut78细胞端粒酶的活性,降低hTERT基因表达,抑制细胞生长增殖及诱导细胞凋亡。

Objective To investigate the effect of small interfering RNA （ siRNA ） directed against human telomerase reverse transcriptase （ hTERT ） on the telomerase activity and proliferation of cutaneous T-cell lymphoma （CTCL） cell line Hut78. Methods Two pairs of siRNAs synthesized with T7 RNA polymerase in vitro transcription were transfected into Hut78 cells by calcium phosphate co-precipitation. The hTERT mRNA expression and telomerase activity were testedby reverse transcription polymerase chain reaction （RT-PCR） and telomeric repeat amplification protocol （TRAP）, respectively. MTT assay was performed to evaluate the cell proliferation. Apoptosis and cell cycles were assessed by using flow cytometry. Results High production of siRNAs were synthesized with T7 RNA polymerase in vitro transcription. The two pairs of siRNAs （ 250 ng） could significantly down-regulate the mRNA expression of hTERT by about 65%, and they suppressed telomerase activity by 68% and 64%, respectively, at 48 h after the transfection. siRNAs could inhibit cell proliferation in a dose-dependent manner; most pronounced inhibition was observed at a dose of 1000 ng. The apoptosis rates of Hut78 cells were 15.86% and 11.10% respectively after transfected with the two pairs of siRNA. No significant changes were found for the above parametres either in calcium phosphate group or in the blank control group. Conclusions The hTERT-siRNA synthesized with T7 RNA polymerase in vitro transcription could suppress the telomerase activity of Hut78 cells, down-regulate the expression of hTERT gene in these cells, inhibit the proliferation, and induce the apoptosis of these cells.