hTERT基因转染显著延长人骨髓间充质干细胞增殖能力的实验研究  

Significant prolongation of reproductive activity and life period in hMSCs by hTERT transfection

作　　者：石东文[1] 代飞[2] 池君[3] 陈希炜[1] 贺伟峰[1] 胡晓红[1] 罗高兴[1] 吴军[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所创伤,烧伤与复合伤国家重点实验室疾病蛋白质组学重庆市市级重点实验室,重庆400038 [2]第三军医大学西南医院骨科,全军矫形外科中心,重庆400038 [3]成都军区昆明总医院耳鼻咽喉科,昆明650032

出　　处：《第三军医大学学报》2007年第16期1562-1565,共4页Journal of Third Military Medical University

基　　金：国家自然科学青年基金(30300090);疾病蛋白质组学重庆市市级重点实验室专项基金(2004-2007)~~

摘　　要：目的构建含有端粒酶逆转录酶(human telom erase reverse transcriptase,hTERT)基因的逆转录病毒,感染人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs),观察外源性hTERT基因在hMSCs中的表达及其对hMSCs端粒酶活性和细胞增殖能力的影响。方法构建pLXSN-hTERT质粒,BamHⅠ酶切和测序鉴定,转染PT67包装细胞,G418抗性筛选获得产病毒阳性细胞克隆。用产病毒细胞珠和hMSCs共培养的方法转染hMSCs,G418抗性筛选。以未转染hM-SCs作为对照,用TRAPEZE方法检测hTERT基因转染hMSCs的端粒酶活性,用流式细胞仪检测细胞周期,并观察转染hMSCs的生长情况。结果成功构建了hTERT基因重组pLXSN-hTERT质粒,建立了产病毒pLXSN-hTER-PT67细胞株。获得了hTERT-hMSCs细胞群,与未转染的hMSCs相比,hTERT-hMSCs细胞群端粒酶活性明显增强,hTERT蛋白阳性表达细胞百分率提高,细胞的生命周期显著延长,细胞传代至46代,形态无明显改变,生长良好。结论逆转录病毒载体介导的hTERT基因转染可增强hMSCs的端粒酶活性,提高hMSCs的增殖能力。Objective To investigate the expression of exogenous human telomerase reverse transcriptase （hTERT） in the human mesenchymal stem cells （hMSCs） and detect the effect of the gene on cellular telomerase activity and proliferative capacity. Methods The plasmid pLXSN-hTERT was constructed and identified by sequencing and enzyme digestion. Then the plasmid was packaged in PT67 and masculine clone cells were screened. The clone was co-cultured with hMSCs. The telomerase activity in the hMSCs was detected by TRAPEZE. The cell cycle was detected by flow cytometry and the growth of hMSCs were observed under inverted microscope. Results A recombinant hTERT-retroviral vector was successfully constructed, and a packaging cell line producing PT67-pLXSN-hTERT virus was established. An hMSCs-TERT cell population was obtained when hMSCs was transfected by means of co-culturing which with the PT67-pLXSN-hTERT virus packaging cells. Compared with the un-infected ones, hTERT-hMSCs cell population acquired stronger telomerase activity, higher hTERT-positive cell ratio, longer cell surviving time and more powerful proliferative capacity, and maintained good growth condition without obvious morphological changes even until passage 46. Conclusion The hTERT gene can be well introduced into hMSCs mediated by retroviral vector, hence prominently increasing the cellular expression of hTERT, telomerase activity and proliferative capacity, and ultimately, delaying cell senescence.

关 键 词：HTERT 逆转录病毒 端粒酶 人骨髓间充质干细胞 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] R331.22[医药卫生—基础医学]