摘要
目的探讨反义人端粒酶(hTERT)基因治疗的可行性。方法构建hTERT基因的反义表达载体,经脂质体介导转染人未分化胃癌细胞系HGC-27,通过Southernblot检测外源反义基因的整合;RT-PCR及DNA测序法检测反义基因的转录;RT-PCR半定量方法检测被封闭目的基因mRNA的转录水平;TRAP及PCRELISA方法检测细胞的端粒酶活性;流式细胞仪检测细胞周期变化。结果外源反义hTERT基因已整合入细胞并获稳定转录,且能显著封闭目的基因转录的mRNA,并显著抑制HGC-27细胞的端粒酶活性,抑制HGC-27细胞的增殖并促进其凋亡。结论端粒酶反义hTERT基因可有效地应用于胃癌的基因治疗。
Objective To construct the retrovirus vector carrying antisense human telomerase reverse transcriptase(hTERT) gene, and to investigate the effect of antisense hTERT transfection on the growth of human gastric cancer cell lines. Methods The cDNA fragment of the hTERT gene was PCR amplified and inserted into the retrovirus vector pLNCX. HGC 27 cells were transfected with the recombinant retrovirus vector with lipofectamine reagent. Southern blot, RT PCR amplification and DNA sequencing were used to detect the conformity and transcription of antisense hTERT gene, and TRAP and PCR ELISA to detect telomerase activity, and flow cytometry to detect cell cycle changes. Results The constructed antisense hTERT expression vector was proved to be the same as designed by restriction endonuclease analysis and sequencing. Exogenous antisense hTERT was successfully transferred into HGC27 cells and obtained stable expression. Antisense hTERT gene significantly blocked the mRNA transcription of the target gene, significantly suppressed the telomerase activity and proliferation of HGC 27 cells and resulted in cells apoptosis. Conclusion Antisense hTERT gene can inhibit hTERT transcription and telomerase activity and result in cell growth arrest, cell cycle arrest and apoptosis, which can be used in gene therapy for gastric cancer.
出处
《中华胃肠外科杂志》
CAS
2004年第5期397-400,共4页
Chinese Journal of Gastrointestinal Surgery
基金
辽宁省自然科学基金资助项目(20022129)
