慢病毒介导稳定过表达β-catenin可促进间充质干细胞的增殖和迁移  

Lentivirus-mediated over-expression of beta-catenin accelerates proliferation and migration of mesenchymal stem cells

作　　者：吴倩[1] 魏亚明 李瑜元[1] 聂玉强[1] 曹燕雯[1] 

机构地区：[1]广州医科大学附属广州市第一人民医院消化科广州市临床医学研究所 [2]输血科,广东省广州市510180

出　　处：《中国组织工程研究》2013年第40期7076-7083,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81070385);留学回国人员科研启动基金(教外司留[2012]940号);广州市重大民生专项基金(201300000100)~~

摘　　要：背景:β-catenin是wnt/β-catenin信号通路中最关键的信号分子,参与调控细胞增殖、分化以及组织自我修复的平衡。目的:建立慢病毒介导的过表达β-catenin基因的大鼠间充质干细胞株,观察β-catenin基因对间充质干细胞增殖和迁移的影响。方法:构建靶向过表达β-catenin基因的慢病毒载体pLV-catenin-RFP,采用人胚肾上皮细胞293T进行包装,收集、浓缩病毒,感染间充质干细胞株,经过嘌呤霉素加压筛选,建立稳定过表达β-catenin基因的间充质干细胞。通过实时荧光定量PCR、细胞生长曲线、MTT实验、Transwell体外细胞迁移实验研究稳定过表达β-catenin基因对间充质干细胞增殖和迁移的影响。结果与结论:成功包装靶向过表达β-catenin基因的慢病毒载体pLV-catenin-RFP,建立稳定过表达β-catenin基因的间充质干细胞。与空载对照pLV-RFP组和正常间充质干细胞组相比,实时荧光定量PCR证实,慢病毒pLV-catenin-RFP组mRNA表达明显增高(P<0.05);MTT和生长曲线显示pLV-catenin-RFP组使细胞倍增时间缩短(P<0.05),增殖能力增加;细胞迁移实验显示pLV-catenin-RFP组使细胞迁移能力增加(P<0.01)。结果可见慢病毒介导稳定过表达β-catenin基因能使间充质干细胞的增殖能力及迁移能力增强。BACKGROUND：β-catenin is the most critical signaling molecule in the Wnt/β-catenin signaling pathway, which is involved in the regulation of cellproliferation, differentiation and tissue self-healing balance. OBJECTIVE：To construct a stableβ-catenin over-expression lentivirus-mediated vector and to transfect mesenchymal stem cells line for investigating its effects on proliferation and migration of mesenchymal stem cells. METHODS：Over-expression vector, PLV-EF1A-catenin-RFP, was constructed and transfected the 293T cellto infect mesenchymal stem cells, and positive cells were selected with puromycin. The up-regulated efficiency of targetingβ-catenin gene at mRNA level was detected by real-time quantitative PCR, the effect on proliferation of mesenchmal stem cellwas assayed by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay and growth curve, and the migration ability was detected by Transwel motility assay. RESULTS AND CONCLUSION：The lentiviral vector targetingβ-catenin gene was constructed successful y, and a stable mesenchymal stem cellline that up-regulatedβ-catenin was established. Real-time quantitative PCR results showed that the expression ofβ-catenin gene was efficiently up-regulated by infecting PLV-EF1A-catenin-RFP （P〈0.05）. The 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay and growth curve showed that celldoubling time was shortened after infected with pLV-EF1A-catenin-RFP （P〈0.05）, indicating that the over-expression of theβ-catenin gene successful y increased the proliferative capability of mesenchymal stem cells. The Transwel assay also showed similar increasing results on the migration ability （P〈0.01）. The lenvivirus-mediated over-expression of theβ-catenin gene can be used to increase the proliferation and migration abilities of the mesenchymal stem cells.

关 键 词：干细胞 干细胞培养与分化 慢病毒 过表达 Β-CATENIN基因 间充质干细胞 增殖 迁移 国家自然科学基金 干细胞图片文章 

分 类 号：R394.2[医药卫生—医学遗传学]