Lentiviral vector mediated hLPP-3 expression in human umbilical cord derived mesenchymal stem cells

doi:10.3969/j.issn.1006-7795.2013.06.018

Abstract

Abstract:

Objective In this article, we isolated and cultured mesenchymal stem cells from umbilical cord(UC-MSCs) and studied their biological characterization in vitro. Furthermore, we transfected UC-MSCs using lentiviral vectors encoding(hLPP-3) gene to detect its expression level in vitro. Methods We first isolated monocytes by collagenase digestion from UC and cultured them. Within 1 week, the cells with spindle shape appeared. Ten days after isolation, the cells could be passaged by trypsinization. The surface markers of cultured cells at passage 3~5 were detected by fluorescence activated cell sorting. At the same time, hLPP-3 gene was sub-cloned into lentiviral vectors and packaged into lentivirus through three plasmids co-transfection method. Then the UC-MSCs were transfected using lentiviral vectors encoding hLPP-3 at different multiplicity of infection(MOI) values. The hLPP-3 mRNA and protein expression level was detected using real-time PCR and Western blotting. Results We isolated and cultured UC-MSCs in vitro successfully. The FACS indicated that the CD90, CD73 and CD105 were positive; however the CD14, CD34, CD45, CD19, HLA-DR, CD11b and CD106 were negative. Real-time PCR showed hLPP-3 mRNA expression level were correlated with the multiplicity of infection(MOI). Western blotting indicated that the over expression of hLPP-3 protein were stable for at least 4 weeks. Conclusion The UC-MSCs could be isolated and cultured in vitro. Its biological characteristics are in accordance with common defined mesenchymal stem cells. Through transfection of UC-MSC by lentiviral vectors encoding hLPP-3, UC-MSCs could express hLPP-3 continuously.

Key words: umbilical cord blood, mesenchymal stem cell, hLPP-3, lentiviral vectors, transduction

CLC Number:

R392.11

LI Xiaobo, GU Jianjuan, ZHAO Chunsong, WANG Shuyan, GUAN Yunqian, CHEN Ling, ZHANG Yu. Lentiviral vector mediated hLPP-3 expression in human umbilical cord derived mesenchymal stem cells[J]. Journal of Capital Medical University, 2013, 34(6): 872-878.

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URL: https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/10.3969/j.issn.1006-7795.2013.06.018

https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/Y2013/V34/I6/872

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