研究逆转录病毒载体介导的反义端粒酶RNA基因对肝癌细胞的抑制作用,探讨通过抑制端粒酶活性治疗肝细胞癌的有效途径。方法: 采用电穿孔法将携带正反义端粒酶RNA基因的逆转录病毒载体导入PT67包装细胞,G418筛选获得稳定产病毒细胞株,收集逆转录病毒上清并感染人肝癌HepG2细胞,G418筛选获得稳定转染细胞克隆并扩增培养,经PCR鉴定后,通过MTT法分别检测转染正反义端粒酶RNA基因组肝癌细胞(HepG2hTREcoRⅠ和HepG2hTRBamHⅠ)以及未转染端粒酶RNA基因组细胞(HepG2)的生长,免疫荧光化学检测肝癌细胞增殖, TRAPPCRELISA法检测细胞的端粒酶活性,流式细胞术检测各组细胞所处的细胞周期及凋亡率。结果: 基因转染后经PCR扩增可于约500 bp处检测到目的基因的表达。转染反义端粒酶RNA基因的HepG2hTRBamHⅠ组细胞生长受到明显抑制；抗中性粒细胞核增殖抗原（PCNA）表达减少;实验组端粒酶活性为(2.31±0.16),较HepG2hTREcoRⅠ组（3.24±0.20）及HepG2组细胞（3.22±0.17）明显下降(P<0.01);流式细胞术检测显示实验组细胞的凋亡率为(9.58±1.38)%,与对照组相比差别具有显著性(P<0.01)；实验组细胞出现G2/M期阻滞。结论: 端粒酶RNA是端粒酶活性表达的一个重要组成部分,通过反义技术下调其表达能够抑制肝癌细胞生长增殖,并诱导其凋亡。

To study the inhibitory effect of retrovirusmediated antisense human telomerase RNA (hTR) gene on hepatocelluar carcinoma, so as to explore an effective way to inhibit telomrerase activity in the treatment of hepatocellular carcinoma. Methods: Sense and antisense hTR gene were transfected into the packaging cell line PT67 by electroporation, and the stablely transfected cells were selected with G418. The recombinant retroviral supernatant was collected and transfected into HepG2 cells. After G418 selection, PCR was used to verify the integration of the hTR gene. Cell growth curves were drawn using MTT assay and the expression of PCNA was determined by immunofluorescence. TRAPPCRELISA was adopted to detect the telomerase activity; cell cycle and apoptosis were evaluated by flow cytometry (FCM).Results: The expression of hTR gene could be amplified in HepG2hTREcoRⅠ and HepG2hTRBamHⅠ cells, but not in untransfected HepG2 cells. The antisense hTR complementary to the template region of telomerase inhibited growth and proliferation of HepG2 cells. The expression of neutrophil proliferating cell nuclear antigen (PCNA) was decreased. Telomerase activity in the antisense hTRtreated group was (2.31±0.16),which was significantly lower than those of the other 2 groups(P<001). FCM showed that the apoptosis rate of the experimental group was (958±138)%, which was significantly different from those of the other 2 groups(P<0.01). G2/M phase blockage was detected in the HepG2hTRBamHⅠ cells. Conclusion: Telomerase RNA is an important component of telomerase; downregulation of its expression through antitechnology can lead to the growth inhibition and apoptosis of hepatocellular carcinoma cells.

山东省医药卫生科研项目（No.CAIDBA3）