神经导向因子Netrin-1基因转染大鼠骨髓间充质干细胞的体外实验  

A study on transfecting neuronal guidance factor Netrin-1 to rat bone mesenchymal stem cells in vitro

作　　者：柯先金[1] 李倩[2] 孙波[2] 张云云[2] 姜婧[2] 曹菁菁[2] 黄素素[2] 丁新生[1] 

机构地区：[1]南京医科大学第一附属医院神经内科,江苏南京210029 [2]南京医科大学附属南京第一医院内分泌科,江苏南京210006

出　　处：《南京医科大学学报（自然科学版）》2011年第3期328-334,共7页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(30971022);江苏省自然科学基金(BK2008471);南京市医学科技发展项目(ZKX08014)资助

摘　　要：目的:将携带增强型绿色荧光蛋白(EGFP)基因标记和Netrin-1基因的腺病毒(Ad5-Netrin-1-CMV-EGFP)转染骨髓间充质干细胞(BMSCs),观察其对BMSCs分泌细胞因子、自身分化和小管形成的影响。方法:体外分离培养大鼠BMSCs,转染病毒后,real-time PCR检测BMSCs表面受体,RT-PCR检测细胞因子的表达,ELISA法检测Netrin-1的表达情况,并通过小管形成实验和Transwell迁移实验观察Netrin-1联合BMSCs对小管形成能力和细胞迁移的影响。结果:腺病毒感染的BMSCs表达多种Netrin-1受体并分泌血管内皮生长因子(VEGF)、成纤维细胞生长因子(bFGF)、脑源性神经营养因子(BDNF)和神经生长因子(NGF)等细胞因子。Netrin-1具有促进HUVEC形成小管能力,但比VEGF差(P<0.05);Netrin-1联合VEGF促进HUVEC形成小管能力比单独Netrin-1或VEGF更强(P<0.05);Transwell迁移实验显示Netrin-1具有与VEGF相当的促进BMSCs迁移能力,单独Netrin-1或VEGF迁移能力不及Netrin-1联合VEGF(P<0.05)。结论:Ad5-Netrin-1-CMV-EGFP成功转染BMSCs并分泌Netrin-1,Netrin-1联合VEGF促进小管形成能力更强,Netrin-1具有促进BMSCs迁移能力,联合VEGF更能促进BMSCs迁移。Objective：To study the effect of transfecting the neuronal guidance factor Netrin-1 and enhanced green fluorescent protein gene（EGFP） to rat bone mesenchymal stem cells（BMSCs） by adenovirus on the cytokine secretion,differentiation and tube formation.Methods：BMSCs were separated and purified in vitro.Surface receptors of adenovirus infected BMSCs and cytokines were detected by real-time PCR or RT-PCR.Tube formation of HUVEC was observed by coculture with BMSCs.BMSCs migration was observed by Transwell experiment.Results：Adenovirus infected BMSCs expressed some kinds of Netrin-1 receptors and secreted vascular endothelial growth factor（VEGF）,basic fibroblast growth factor（bFGF）,brain derived neurophic factor（BDNF） and nerve growth factor（NGF）.BMSCs cultured with Netrin-1 and VEGF markedly increased the tube formation of HUVEC and BMSCs migration,compared with Netrin-1 or VEGF alone（P 〈 0.05）.Conclusion：BMSCs successfully infected with Ad5-Netrin-1-CMV-EGFP can express EGFP and Netrin-1.Netrin-1 has the ability to accelerate tube formation and BMSCs migration and VEGF can enhance the effect when used together.

关 键 词：NETRIN-1 骨髓间充质干细胞 小管形成 Transwell迁移 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]