稳定过表达生长分化因子5基因大鼠脂肪干细胞系的建立

摘要

目的:探讨慢病毒介导的稳定过表达生长分化因子5(GDF-5)基因大鼠脂肪干细胞(ASCs)的构建条件和方法.方法:取大鼠腹股沟脂肪垫组织,采用Ⅰ型胶原酶消化贴壁法分离培养大鼠ASCs.倒置相差显微镜观察细胞形态,CCK-8法测定细胞生长曲线,流式细胞仪鉴定细胞表型.制备带GDF-5/GFP融合基因的慢病毒载体系统,探索不同感染复数(MOI=1、5、10、20、40、60、80、100)的转染效率,选择最佳MOI,采用流式细胞仪检测转染效率.对转染细胞行流式细胞筛选,测定筛选后转染细胞的阳性率.筛选出的细胞行细胞爬片,DAPI染色,形态学上进一步验证细胞阳性率;并采用CCK-8法检测转染后细胞活力.结果:成功培养大鼠ASCs,流式细胞免疫表型鉴定:间充质干细胞表面抗原(CD90、CD29、CD44、CD105)表达阳性,造血细胞表面抗原(CD45、CD34)和骨髓干细胞表面抗原(CD106)表达阴性.成功构建GDF-5过表达慢病毒载体系统,慢病毒转染大鼠ASCs最佳MOI为40,转染率为65%.采用GFP荧光流式细胞筛选技术筛选后阳性率可提高至96%.CCK-8法显示,转染后细胞活力、生长曲线与未转染细胞无明显差异.结论:胶原酶消化法可成功培养大鼠ASCs,流式细胞筛选技术可显著提高转染细胞阳性率,且对细胞系活力无显著影响,转染细胞传代5代仍可稳定过表达.%Objective:To explore the optimal multiplicityof infection (MOI) construction conditions and methods of lentivirus-mediated GDF-5 gene over-expressed rat adipose stem cells (ASCs-GDF-5).Methods:Rat ASCs were isolated and cultured using collagenase digestion method.The cell morphology was observed with the growth curve being tested.Besides, the cell phenotypes were identified.Lentiviral vector system with GDF-5/GFP chimeric gene was prepared and the infection efficiency was explored under series of MOI (1, 5, 10, 20, 40, 60, 80, 100).The optimal MOI was determined and the infection efficiency was tested using FCM.High-purified ASCs-GDF-5 were obtained through fluorescence activated cell sorting with FCM.The positive rate of infected cells was verified further with DAPI staining.The viability of infected cells was evaluated with CCK-8 assay.Results:Rat ASCs were successfully isolated and cultured.The markers (CD90, CD29, CD44, CD105) expressed in mesenchymal stem cell were positive in cultured cells.In contrast, the hematopoietic cell surface antigens (CD45, CD34) and bone marrow stem cell surface antigen (CD106) were negative.GDF-5 gene over-expressed lentiviral vector system was successfully constructed.The optimal MOI was 40, with the infection rate of 65%.The positive rate of infected cells was increased to 96% through fluorescence activated cell sorting using FCM.There was no significant difference in the viability and growth curve between infected and non-infected cells with CCK-8 assay.Conclusions:Rat ASCs can be cultured using collagenase digestion method.Without significant effect on cell viability, the positive rate of infected cells can be significantly increased through fluorescence activated cell sorting using FCM.