逆转录病毒方法建立小鼠诱导性多能干细胞的实验研究  

Research of Mouse Induced Pluripotent Stem Cell Created by Retrovirus Approach

作　　者：单威[1] 余勤[1] 王标[1] 刘丽珍[2] 刘森泉[2] 付珊[2] 刘伟[1] 周丽萍[1] 

机构地区：[1]浙江中医药大学生物工程学院,杭州310053 [2]浙江大学医学院附属第一医院

出　　处：《浙江中医药大学学报》2013年第2期111-115,共5页Journal of Zhejiang Chinese Medical University

基　　金：浙江省科技计划项目(2009C14011);国家自然科学基金(81270566)~~

摘　　要：[目的]采用逆转录病毒法转染P2代ICR的小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF),建立小鼠诱导性多能干细胞(inducedpluripotent stem cell,iPS cell,iPS细胞)。[方法]本实验采用293T细胞进行逆转录病毒包装,将分别携带来源于人的Oct3/4、Sox2、c-Myc、Klf4转录因子的4种逆转录病毒液共转染ICR小鼠的MEF,并在转染过程中添加维生素C、丙戊酸钠来提高小鼠iPS细胞的诱导效率,通过碱性磷酸酶染色、RT-PCR方法检测建立的细胞株。[结果]转染的MEF在1周左右时间出现明显克隆,克隆传代的细胞经碱性磷酸酶染色呈紫红色,RT-PCR检测结果显示细胞株中的Oct3/4、Sox2、c-Myc、Klf4、Nanog 5种多能性相关的基因都得到明显表达。[结论]转染的MEF在1周左右时间出现明显克隆,小鼠iPS细胞的一些表面标志如碱性磷酸酶得到表达,Oct3/4、Sox2、c-Myc、Klf4、Nanog 5种多能性相关基因也都得到明显表达,表明已经建立小鼠iPS细胞。[Objective] To create the mouse induced pluripotent stem cell（miPSC） by thansfecting the ICR mouse embryonic fibroblast（MEF） of passage 2 through the retrovirus approach in order to provide the theoretical basis for clinical application.[Methods] The research applied the retrovirus containing the human cDNA,Oct4,Sox2,Klf4,and c-Myc packaged by 293T cell to infect the ICR MEF,in the process of which we used the Vitamin C and Valproic acid sodium to improve the efficiency of inducement.We identified the miPSC by alkaline phosphatase staining and RT-PCR.[Results] The infected MEF apparently changed to iPSC clones and the amplified colonies displayed purple-red color by alkaline phosphatase staining.The pluripotency marker genes Oct3/4,Sox2,c-Myc,Klf4 and Nanog were highly expressed detected by RT-PCR.[Conclusion] The infected MEF apparently changed to iPSC clones and the alkaline phosphatase,the pluripotency marker genes Oct3/4,Sox2,c-Myc,Klf4,Nanog also showed high expression,which suggested we had created the miPSC successfully.

关 键 词：IPS细胞 MEF 逆转录病毒 

分 类 号：R331[医药卫生—人体生理学]