逆转录病毒介导外源基因高效感染大鼠骨髓间充质干细胞  

Retroviral-mediated high efficient expression of target gene in expanded rat mesenchymal stem cells

作　　者：郑晴雯[1] 董晓先[1] 彭燕[1] 董伟华[1] 梁仲培[1] 何慧华[1] 周萍[1] 刘金保[1] 

机构地区：[1]广州医学院病理生理学教研室,广东广州510182

出　　处：《中国现代医学杂志》2005年第22期3424-3427,3431,共5页China Journal of Modern Medicine

基　　金：国家863计划基金资助项目(No:2002AA204011);广州市科技攻关引导项目(No:2003Z3-E0012)

摘　　要：目的通过逆转录病毒系统将小鼠同源盒基因Sox1转移至大鼠骨髓间充质干细胞(rMSC),检测外源基因的表达,感染效率及感染时间。方法常规分子生物学亚克隆技术构建重组逆转录病毒载体,转染至包装细胞株PT67,制备含目的基因的重组逆转录病毒液,感染rMSC,并用带EGFP表达盒的pLEGFP-N1载体的逆转录病毒系统作为平行对照,评估感染率;使用Westernblot以及免疫组织化学的方法检测携带目的基因的重组逆转录病毒在rMSC中的感染和表达情况。结果本实验成功地制备了pLXSN-Sox1重组逆转录病毒载体并制备出高滴度病毒颗粒;将病毒上清感染rMSC,EGFP阳性细胞为21%,感染时间持续20d,而对照脂质体转染法EGFP阳性细胞仅为3%。免疫组化及Westernblot显示用重组逆转录病毒液感染的rMSC可表达外源基因产物Sox1蛋白。结论采用逆转录病毒介导的方法可以将外源性基因高效转移至rMSC中,并有外源性基因的稳定表达。[Objective] Investigate the expression and infection efficiency,infection time of retroviral delivery of genes in vitro by delivering target gene mouse homeobox gene Soxl into mesenchymal stem cells （rMSC） with retroviral system, [Methods] The recombinant retroviral plasmid DNA carrying the Soxl gene was constructed by subclone technique, then transfected to PT67 cells to package recombinant retroviral particles, rMSC was infected with the recombinant retroviral particles, pLEGFP-N1 containing enhanced green fluorescent protein gene （EGFP） expression cassette was used as a parallel matched control, Infection and expression efficiency were assessed by detecting the expression of EGFP, analysis of Western blot and immunohistochemistry. [Results] The recombinant retroviral vector pLXSN-Soxl and made the high efficient expression of the retroviral particles Retro-Soxl, When infected by the retroviral particles, 21% of the rMSC was positive to EGFP and last for 20 days, Only 3% of the rMSC was positive for EGFP in comparison group used routine method of lipofectamin mixed with plasmid DNA, Immunohisto-chemistry and Western blot showed that target gene Soxl was expressed in infected rMSC, [Conclusion] Recombinant retrovirus high-effectively mediated gene transfer in expanded rMSC, with the stable expression of target gene.

关 键 词：同源盒基因 逆转录病毒载体 基因转移 骨髓间充质干细胞 绿色荧光蛋白 

分 类 号：Q78[生物学—分子生物学]