天津医药

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慢病毒载体介导apelin基因转染人脐带间充质干细胞的实验研究

王力1,张宁坤2,高连如2,徐小红2,郑楠2,朱智明2   

  1. 1. 第二军医大学海军临床医学院,中国人民解放军海军总医院心脏中心
    2. 中国人民解放军海军总医院心脏中心
  • 收稿日期:2013-07-31 修回日期:2013-08-26 出版日期:2013-12-15 发布日期:2013-12-15
  • 通讯作者: 王力

Transfection of Lentivirus Vector Containing Apelin Gene into Umbilical Cord Mesenchymal Stem Cells in Vitro

WANG Li 1,ZHANG Ning kun2,GAO Lian ru2,XU Xiao hong2,ZHENG Nan 2,ZHU Zhi ming2   

  1. 1. Clinical College of Navy Medicine, Second Military Medical University Department of Cardiology, Navy General Hospital
    2. Department of Cardiology, Navy General Hospital
  • Received:2013-07-31 Revised:2013-08-26 Published:2013-12-15 Online:2013-12-15
  • Contact: WANG Li

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摘要：

【摘要】  目的  构建带有荧光标记基因增强型绿色荧光蛋白（EGFP）和apelin基因的重组质粒pUbi-apelinFLAG-pSV40-EGFP并进行慢病毒包装，探讨其转染人脐带间充质干细胞的最佳感染复数（MOI）值及目的基因表达情况。  方法  化学合成目的基因片段并扩增。采用In-Fusion技术将酶切后的目的基因片段与线性质粒载体连接，转化入感受态DH5α细胞中后筛选阳性克隆并进行测序。重组质粒慢病毒载体转染293T细胞，包装慢病毒并测定滴度。将重组质粒慢病毒按不同MOI值转染人脐带间充质干细胞，根据转染效率得到最佳MOI值，并采用Real-timePCR及Western blot方法检测目的基因表达情况。 结果  通过PCR扩增获得酶切位点碱基修饰后的大小约284bp的目的基因片段，与慢病毒质粒载体连接后形成pUbi-apelin-FLAG-pSV40-EGFP重组质粒，测序结果与预期完全符合，并成功包装慢病毒颗粒。最佳MOI值为20，转染效率为（90.32±3.61）%。慢病毒载体能高效转染人脐带间充质干细胞且2周内持续稳定上调目的基因mRNA及蛋白的表达。  结论  重组质粒慢病毒载体pUbi-apelin-FLAGpSV40-EGFP可有效转染人脐带间充质干细胞，并可持续高表达apelin基因

关键词: 间充质干细胞, 慢病毒表达载体, 转染, 基因表达, apelin

Abstract:

[Abstract]   Objective  To construct a recombinant plasmid pUbi-apelin-FLAG-pSV40-EGFP and package with
lentivirus to co-express enhanced green fluorescent protein (EGFP) and apelin, and to investigate optimal multiple of infection (MOI) to transfect human umbilical cord mesenchymal stem cells (hUCMSCs) and expression of target gene.  Methods   he apelin gene was chemically synthesized and amplified by polymerase chain reaction (PCR), and which was inserted into inear plasmid vector. The gene fragment and linear plasmid vector were connected by In-Fusion technology after enzyme digestion and transformed into competent DH5αcells. The positive clones of lentiviral expression vector were obtained after creening and followed by sequencing. The lentiviral vector was used to transfect293T cells and package virus, and then the irus titers were determined. HUCMSCs were transfected with lentivirus vector in vitro via different values of MOI. The transfection efficiency was obtained according to the optimal MOI. The expression of target gene was detected by RT-PCR and estern blot assay.  Results   A284bp target gene fragment with the restriction sites was obtained by PCR and connected to he lentiviral vector. The positive clones of lentiviral expression vector were corresponded to the expected result. The lentiviral particles were successfully packaged. HUCMSCs could be transfected by the lentivirus vector with high efficiency. The RNA and protein levels of target gene were stably up-regulated within2weeks.  Conclusion   The lentivirus vector pUbiapelin-FLAG-pSV40-EGFP can transfect apelin gene into hUCMSCs with high efficiency. The infected cells can express igh levels of apelin gene in two weeks.

Key words: mesenchymal stem cell, Lentiviral vector, transfection, gene expression, apelin