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

中国儿童保健杂志 ›› 2014, Vol. 22 ›› Issue (10): 1018-1021.

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

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

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

收稿日期:2014-02-13 发布日期:2014-10-10 出版日期:2014-10-10
通讯作者: 倪毓辉,E-mail:niyuhui@hotmail.com
作者简介:杨蕾(1987-)，女，博士在读，研究方向为儿童肥胖症。
基金资助:
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)

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

YANG Lei^1,2,JI Chen-bo^1,2,SHI Chun-mei^1,2,CHEN Ling^1,2,PANG Ling-xia^1,2,XIA Li^1,2,GUO Xi-rong^1,2,NI Yu-hui^1,2

1 Institute of Pediatrics,Nanjing Medical University,Nanjing,Jiangsu 210029,China;
2 Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University,Nanjing,Jiangsu 210004,China

Received:2014-02-13 Online:2014-10-10 Published:2014-10-10
Contact: NI Yu-hui,E-mail:niyuhui@hotmail.com

摘要/Abstract

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，36 h开始观察荧光标记，72 h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。

关键词: miR-1908, 慢病毒, 人脂肪前体细胞

Abstract: Objective To construct a recombinant lentiviral vector carrying miR-1908 and validate its infection efficiency 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 increased nearly two folds in infected cells. Conclusion It has been successfully constructed that the lentiviral vector containing 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

Key words: miR-1908, lentivirus, human preadipocytes

中图分类号:

R394

杨蕾，季晨博，史春梅，陈玲，庞玲霞，夏黎，郭锡熔，倪毓辉. 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[J]. 中国儿童保健杂志, 2014, 22(10): 1018-1021.

YANG Lei,JI Chen-bo,SHI Chun-mei,CHEN Ling,PANG Ling-xia,XIA Li,GUO Xi-rong,NI Yu-hui. Construction of a recombinant lentiviral vector carrying miR-1908 and validation of its transduction efficiency in human pre-adipocytes[J]. journal1, 2014, 22(10): 1018-1021.