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

作　　者：李晓波[1,2] 顾建娟 赵春松[2] 王淑艳[2] 关云谦[2] 陈凌[2] 张愚[2] 

机构地区：[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

出　　处：《首都医科大学学报》2013年第6期872-878,共7页Journal of Capital Medical University

基　　金：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)~~

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-3mRNA的转录水平和蛋白表达量。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 CDg0, CD73 and CD105 were positive; however the CD14, CD34, CD45, CD19, HLA-DR, CDllb 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 l）iological characteristics are in accordance with common defined mesenchymal stein cells. Through transfection of UC-MSC by lentiviral vectors encoding hLPP-3, UC-MSCs could express hLPP-3 continuously.

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 

分 类 号：R392.11[医药卫生—免疫学]