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

Establishment of stable GDF-5 gene over-expressed rat adipose stem cells

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。

Objective：To explore the construction conditions and methods of lentivirus-mediated GDF-5gene overexpressed 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-5were 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-8assay.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-5gene 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-8assay.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.