【摘要】 背景与目的:抑制端粒酶活性可以抑制细胞端粒延长,进而抑制永生细胞的增殖。为了探讨以端粒酶为靶的肺癌基因治疗可能性,本实验观察反义人端粒酶逆转录酶组分(humantelomerasereversetranscriptase,hTERT)cDNA对A549肺癌细胞的端粒酶活性和细胞增殖的抑制作用。方法:RT-PCR扩增hTERTmRNA5’起始端835bp的cDNA,分别正向和反向插入到逆转录病毒表达载体pLXSN质粒中,转染包装细胞PT67后获得重组病毒,病毒感染肺癌A549细胞。Westernblot检测hTERT蛋白表达,TRAP法检测端粒酶活性,倒置显微镜观察细胞形态变化和绘制细胞生长曲线了解细胞增殖情况,流式细胞仪和DNA片段电泳观察凋亡情况。结果:反义hTERT作用肺癌A549细胞后hTERT蛋白表达下降,端粒酶活性被抑制,细胞增殖受到抑制并出现明显的细胞凋亡。结论:反义hTERT对肺癌细胞A549端粒酶活性具有明显的抑制作用,抑制细胞生长,促进细胞凋亡,hTERT有可能成为肺癌基因治疗靶点。更多还原

【Abstract】 BACKGROUND &OBJECTIVE: Inhibition of telomere length can be achieved through suppression of telomerase activity, which may result in the inhibition of immortal cell proliferation. In order to explore the possibility of the telomerase as a target for lung cancer therapy, we investigated the effects of anti sense human telomerase reverse transcriptase (hTERT) on telomerase activity and cell proliferation of A549 lung cancer cell line. METHODS: The anti sense hTERT cDNA, an 835 bp in the 5′region of hTERT mRNA was amplified by reverse transcription polymerase chain reaction (RT PCR), before cloning into pLXSN retroviral vector in sense and anti sense orientations. A549 cells, a human lung cancer cell line, were infected with recombinant virus obtained after transfection into packaging cell PT67. The expression of hTERT protein was determined by Western blot analysis. The telomerase activity was measured by telomerase repeat amplification protocol (TRAP). The cell proliferation was depicted by cell morphology under inverted microscopy as well as cell growth curve. Apoptosis was analyzed by flow cytometry and DNA electrophoresis. RESULTS: Compared with sense hTERT transduction, hTERT expression and telomerase activity significantly decreased in A549 cells after anti sense hTERT transduction. The cell proliferation was markedly inhibited with evidence of apoptosis. CONCLUSION: Anti sense hTERT exhibited significant inhibition of telomerase activity and cell proliferation, in addition to acceleration of apoptosis. This implied the possibility of hTERT as the potential target for gene therapy of lung cancer.更多还原