人骨形态发生蛋白-9基因修饰SD大鼠骨髓间充质干细胞的建立研究  

Establishment of bone morphogenetic protein-9 gene modified SD rat′s bone marrow mesenchymal stem cells

作　　者：向晓波[1] 周艳萌[2] 蒋练[1] Xiang Xiaobo Zhou Yanmeng Jiang Lian(Department of Stomatological Surgery,Affiliated Stomatology Hospital of Zunyi Medical College ,Zunyi, Guizhou 563000, China Department of Microbiology and Immunology,Zunyi Medical University,Zunyi, Guizhou 563000, China)

机构地区：[1]遵义医学院附属口腔医院口腔外科,贵州遵义563000 [2]遵义医学院微生物与免疫学实验室,贵州遵义563000

出　　处：《现代医药卫生》2017年第6期801-804,共4页Journal of Modern Medicine & Health

基　　金：贵州省科研基金资助项目(黔科合J字LKZ[2010]03号)

摘　　要：目的建立人骨形态发生蛋白-9(BMP-9)基因转染SD大鼠骨髓间充质干细胞(BMSCs),并观察其在细胞内的表达。方法采用全骨髓细胞贴壁法分离出BMSCs,利用流式细胞仪检测CD44、CD90、CD45、CD34细胞表面标记物;通过脂质体转染法将p Double Ex-EGFP-BMP-9质粒、p Double Ex-EGFP质粒转染入SD大鼠BMSCs,利用荧光显微镜、荧光定量PCR及蛋白质印迹法检测BMP-9的转染效果。结果全骨髓贴壁培养法成功分离大鼠BMSCs,传代培养至第3代可获得较高纯度,CD44-FITC的阳性表达率为92.35%,CD90-FITC的阳性表达率为91.76%,CD34-PE的阳性表达率为2.68%,CD45-PE的阳性表达率为4.13%;荧光显微镜和倒置显微镜下观察,BMP-9基因修饰后细胞形态未发生变化;PCR检测发现p Double Ex-EGFP-BMP-9转染组中大量表达BMP-9基因,是p Double Ex-EGFP组的378 517.88倍;Western blotting结果显示,p Double Ex-EGFP-BMP-9组中大量表达BMP-9蛋白,p Double Ex-EGFP未见表达。结论全骨髓贴壁培养法可以成功分离BMSCs,BMP-9基因可以通过脂质体转染的方法转染入SD大鼠的BMSCs中并得到成功表达。Objective To establish the gene BMP-9-transfected SD rat bone marrow mesenchymal stem cells（BMSCs）and verify the overexpression.Method The BMSCs were isolated from the adherent whole bone marrow cells and the cell surface markers including CD44,CD90,CD45 and CD34 were detected by flow cytometry.p Double Ex-EGFP-BMP-9 plasmid and p Double Ex-EGFP plasmid were transfected into rat BMSCs and the efficiency of the transfection was analyzed by fluorescence micro-scope,quantitative RT-PCR and Western blot.Results BMSCs were successfully isolated by adherent culture of whole bone marrow for three generations.The positive expression rate of CD44-FITC in those cells was 92.35%,the positive expression rate of CD90-FITC was 91.76%,the positive rate of CD34-PE was 2.68%,and the positive rate of CD45-PE was 4.13%.NO morphological change was observed after BMP-9 gene modification in rat BMSCs.The BMP-9 gene expression in the p Double Ex-EGFP-BMP-9group is 378 517.88 times more than that of p Double Ex-EGFP group in RT-PCR test.Western boltting assay showed that the protein expression of BMP-9 in the p Double Ex-EGFP-BMP9 group was obviously higher than that in the the p Double Ex-EGFP group.Conclusion BMSCs could be successfully obtained by adherent culture of whole bone marrow.BMP-9 could be successfully over-expressed in rat BMSCs by liposome transfection.

关 键 词：骨髓 间质干细胞 转染 大鼠 Sprague-Dawley 骨形态发生蛋白质类 基因 

分 类 号：R341[医药卫生—基础医学]