人miR-1908慢病毒载体构建及在人脂肪前体细胞中的表达验证  

Construction of a recombinant lentiviral vector carrying miR-1908 and validation of its transduction efficiency in human pre-adipocytes

作　　者：杨蕾[1,2] 季晨博[1,2] 史春梅[1,2] 陈玲[1,2] 庞玲霞[1,2] 夏黎[1,2] 郭锡熔[1,2] 倪毓辉[1,2] 

机构地区：[1]南京医科大学儿科研究所,江苏南京210029 [2]南京医科大学附属南京妇幼保健院儿童保健科,江苏南京210004

出　　处：《中国儿童保健杂志》2014年第10期1018-1021,共4页Chinese Journal of Child Health Care

基　　金：国家自然科学基金(81100618);江苏省自然科学基金(BK2011107);南京市科技发展计划(201104013)

摘　　要：目的构建人miR-1908慢病毒表达载体,包被病毒并检测其在人脂肪前体细胞中过表达效果。方法以人脂肪细胞基因组DNA为模板,PCR高保真扩增包含miR-1908区域上下游各100bp左右的基因组DNA,亚克隆入慢病毒表达载体,与包装质粒共转染HEK-293T细胞,包装病毒,收取病毒上清,纯化后感染人脂肪前体细胞,36h开始观察荧光标记,72h收取细胞,抽提RNA,Realtime PCR检测慢病毒感染下miR-1908的相应表达量。结果成功构建人miR-1908慢病毒表达载体,包装获得的病毒感染人脂肪前体细胞的效率可达到70%以上,miR-1908过表达水平可接近2倍。结论本研究成功构建了人miR-1908慢病毒过表达载体,包被的慢病毒可以在人脂肪前体细胞中实现过表达效果,为后续功能研究奠定了基础。Objective To construct a recombinant lentiviral vector carrying miR-1908 and validate its infection efficien- cy in human pre-adipocytes. Methods The minigene fragments of miR-1908 was amplified from genomic DNA of human pre-adipocytes by PCR, then cloned into a lentivirus expression vector. After sequencing validated exactly, the expression plasmid and packaging plasmids were co-transfected into HEK-293T cells to produce lentivirus;The supernatant containing lentiviral particles was used to infect human pre-adipocytes, and its infection efficiency and miR-1908 overexpression level were respectively tested by fluorescent observation and real-time quantitative PCR. Results miR-1908 lentiviral vector was constructed successfully, and infection efficiency in human pre-adipocytes reached over 70%, the expression level in- creased nearly two folds in infected cells. Conclusion It has been successfully constructed that the lentiviral vector contai- ning miR-1908 with high overexpression in infected human pre-adipocytes,which will be useful to the future studying the function of miR-1908 in human pre-adipocytes.

关 键 词：miR-1908 慢病毒 人脂肪前体细胞 

分 类 号：R394[医药卫生—医学遗传学]