反义hTERT体外诱导肺癌细胞凋亡的研究  

A study on cell apoptosis of lung cancer induced by antisense hTERT in vitro

作　　者：尹为华[1] 马雅[1] 余光银[1] 李若馨[2] 孙来保[2] 

机构地区：[1]北京大学深圳医院病理科,广东深圳518036 [2]深圳市儿童医院

出　　处：《中国基层医药》2005年第6期726-728,i004,共4页Chinese Journal of Primary Medicine and Pharmacy

摘　　要：目的研究反义hTERT基因体外诱导肺癌细胞凋亡。方法体外通过SuperFect(QIAGEN)转染正、反义hTERT真核表达载体至人肺腺癌细胞株GLC82,再经过G418及PCR筛选鉴定获得分别转入了正、反义hTERT载体的抗性克隆细胞GLC82s和GLC82as。然后采用MTT法、双层软琼脂克隆形成试验、流式细胞术观察和分析了反义hTERT对肺癌细胞体外生长增殖活力的影响及是否能诱导瘤细胞的凋亡;同时还运用实时荧光定量RTPCR技术及TRAP银染法对各组细胞内源性hTERTmRNA的表达情况及端粒酶的活性进行了检测。结果当各组细胞传至第25代后,与GLC82、GLC82s比较,GLC82as细胞的生长速度和集落形成能力明显地减慢和降低,同时伴有凋亡率的显著增加;与空白对照、正义hTERT组相比,反义hTERT能显著地降低GLC82细胞内源性hTERTmRNA的表达(P<0.01)和下调端粒酶活性。结论反义hTERT体外确实能明显地诱导肺癌细胞的凋亡及抑制瘤细胞的生长增殖能力。Objective To study the cell apoptosis of lung cancer induced by antisense hTERT in vitro.Methods The sense and antisense hTERT eukaryotic expression vectors were transfected into lung cancer cell line GLC-82 using the SuperFect transfection reagent(Qiagen) according to the manufacturer's instructions,then the GLC-82-s and GLC-82-as of G418-resistant colonies were obtained with G418 and identified for the presence of hTERT insert by PCR.After that,MTT assay,soft agar colony formation assay and flow cytometry were adopted to analyze whether the proliferation capacity of lung cancer cell were effected in vitro and the tumor cells can be induced to apoptosis by antisense hTERT.Meanwhile,we had also detected the endogenous hTERT mRNA expression and telomerase activity by quantitative real-time RT-PCR and TRAP-silver staining assay in each group cells.Results After 25 passages of three group cells,a 7-day cell growth curve and the numbers(size) of soft agar colony formation showed the proliferation rates and the anchorage-independent growth ability in GLC-82-as were significantly decreased and reduced,compared with GLC-82 and GLC-82-s.But there was a significant increase in apoptosis percentage of GLC-82-as by flow cytometry,compared with control groups.Antisense hTERT could remarkably reduce the endogenous hTERT mRNA expression(P<0.01) and down-regulate telomerase activity in GLC-82,compared with blank control and sense hTERT groups.Conclusion Antisense hTERT can obviously induce cell apoptosis and inhibit the growth/proliferation of lung cancer cell in vitro.

关 键 词：反义HTERT 肺癌 细胞凋亡 流式细胞术 逆转录聚合酶链反应 

分 类 号：R734.2[医药卫生—肿瘤]