反义核酸hTERT对大肠癌细胞的抑制作用

Construction of antisense hTERT retroviral vector and its inhibition on the proliferation of HT29 colorectal cancer cells

目的 :构建人端粒酶逆转录酶组分 (humantelomerasereversetranscriptase ,hTERT)反义cDNA逆转录病毒载体 ,观察其对大肠癌细胞HT2 9端粒酶活性和细胞增殖的抑制作用 ,探讨以hTERT为靶的大肠癌基因治疗的可行性。方法 :RT PCR扩增hTERTmRNA 5′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PT6 7后获得重组病毒 ,病毒感染大肠癌HT2 9细胞。处理前后采用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 ,绘制细胞生长曲线 ,流式细胞仪和DNA片段电泳观察凋亡情况。结果 :反义hTERT作用大肠癌HT2 9细胞后hTERT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对大肠癌细胞HT2 9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有可能成为大肠癌基因治疗靶点。

Objective: To construct the antisense human telomerase reverse transcriptase (hTERT) cDNA retroviral vector, investigate the inhibition of telomerase activity and celluler proliferation in colorectal cancer cells HT29 by it treatment. and explore the possibility of the hTERT as a target for colorectal cancer gene therapy. Methods:We used RT PCR amplify the hTERT mRNA 5′ region 835bp fragment, and cloned the hTERT fragment into pLXSN retroviral vector in sense and antisense orientations. The replication deficient retroviruses were by obtained transfecting the packing cells of PT67, and then infecting the HT29 colorectal cancer cells. The hTERT protein expression was detected by Western blot, telomerase activity was determined by telomerase repeat amplification protocol (TRAP), cell proliferation was investigated by invert microscope and growth curve, and the apoptosis was characterized by flow cytometry and DNA electrophoresis. Results: Compared with the sense hTERT transduced HT29, the hTERT protein expression, telomerase activity and cell proliferation of HT29 were significantly inhibited and apoptosis occurred after antisense hTERT transduction. Conclusion: The transduction of antisense hTERT can significantly inhibit the hTERT expression, suppress telomerase activity, arrest the cell growth and accelerate apoptosis in HT29. hTERT is a potential target for colorectal cancer gene therapy.