慢病毒载体介导的人磷脂磷酸水解酶3基因在人脐带间充质干细胞中的表达

doi:10.3969/j.issn.1006-7795.2013.06.018

首都医科大学学报 ›› 2013, Vol. 34 ›› Issue (6): 872-878.doi: 10.3969/j.issn.1006-7795.2013.06.018

慢病毒载体介导的人磷脂磷酸水解酶3基因在人脐带间充质干细胞中的表达

李晓波^1,2, 顾建娟³, 赵春松², 王淑艳², 关云谦², 陈凌², 张愚²

1. 扬州大学苏北人民医院神经内科, 江苏扬州 225001;
2. 首都医科大学宣武医院细胞生物学研究室, 北京 100053;
3. 扬州大学苏北人民医院妇产科, 江苏扬州 225001

收稿日期:2013-06-17 出版日期:2013-12-21 发布日期:2013-12-13
通讯作者: 关云谦 E-mail:guanyunqian@hotmail.com
基金资助:
国家自然科学基金（81272804；81071011）；教育部重点项目（210003）；江苏省卫生厅资助项目（H201049）；北京市教育委员会科技计划项目（KZ201310025024；KM201010025015）；北京市科技新星计划（2009B22）。

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

LI Xiaobo^1,2, GU Jianjuan³, ZHAO Chunsong², WANG Shuyan², GUAN Yunqian², CHEN Ling², ZHANG Yu²

1. Department of Neurology, Northern Jiangsu People's Hospital, Yangzhou University, Yangzhou, 225001, Jiangsu Province, China;
2. Department of Cell Biology, Xuanwu Hospital, Capital Medical University, Beijing 100053, China;
3. Department of Gynaecology and Obstetrics, Northern Jiangsu People's Hospital, Yangzhou University, Yangzhou 225001, Jiangsu Province, China

Received:2013-06-17 Online:2013-12-21 Published:2013-12-13
Supported by:
This study was supported by the National Natural Science Foundation of China(81272804;81071011);Key Project of Chinese Ministry of Education(210003);Project of Jiangsu Province Department of Health(H201049);Beijing Municipal Education Commission Science and Technology Program(KZ201310025024;KM201010025015);Beijing Nova Program of Science and Technology(2009B22).

摘要/Abstract

摘要：

目的体外分离和培养人脐带间充质干细胞（umbilical cord mesenchymal stem cell，UC-MSCs），并将含有人磷脂磷酸水解酶3（human lipid phosphate phosphatases 3，hLPP3）的慢病毒载体转染UC-MSCs，探讨hLPP-3在UC-MSCs中的表达。方法从脐带中分离单细胞，经细胞形态学、流式细胞仪（FACS）检测、细胞分化潜能判定，鉴定UC-MSCs的生物学特性。同时将hLPP-3基因克隆入慢病毒载体，包装出病毒上清，以不同拷贝数转染UC-MSCs，用免疫印迹和实时定量PCR技术分别测定不同转染组hLPP-3的蛋白及mRNA表达水平。结果成功在体外分离和培养了UC-MSCs，并诱导其向脂肪、骨、软骨细胞分化。流式细胞仪检测结果显示脐带MSC表达CD90、CD73及CD105，而不表达CD14、CD34、CD45、CD19、HLA-DR、CD11b及CD106蛋白。用含hLPP-3-GFP融合基因的慢病毒载体转染UC-MSCs，实时定量PCR检测发现，hLPP-3的mRNA转录水平与转染拷贝数有关，转染拷贝数越高，hLPP-3的表达量越高；转染后1个月，免疫印迹发现hLPP-3-GFP依然维持高表达。结论成功地在体外分离和培养了UC-MSC，并用含有hLPP-3基因的慢病毒载体转染UC-MSCs，通过转染拷贝数在一定水平上调控了UC-MSC中hLPP-3 mRNA的转录水平和蛋白表达量。

关键词: 脐带, 间充质干细胞, 胶质源性神经营养因子, 慢病毒载体, 转染

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

中图分类号:

R392.11

李晓波, 顾建娟, 赵春松, 王淑艳, 关云谦, 陈凌, 张愚. 慢病毒载体介导的人磷脂磷酸水解酶3基因在人脐带间充质干细胞中的表达[J]. 首都医科大学学报, 2013, 34(6): 872-878.

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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慢病毒载体介导的人磷脂磷酸水解酶3基因在人脐带间充质干细胞中的表达

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

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