人骨形态发生蛋白-2基因转染兔骨髓基质干细胞及其表达  

Expression and mechanism of cDNA of human bone morphogenetic protein 2 via eukaryotic expression vector in rabbit mesenchymal stem cells

作　　者：邹德波[1] 周东生[1] 王永会[1] 王来城[1] 武兴国[1] 黄涛[1] 

机构地区：[1]山东省立医院骨科,济南250021

出　　处：《中华创伤骨科杂志》2006年第2期148-152,共5页Chinese Journal of Orthopaedic Trauma

基　　金：山东省科学技术发展计划项目(023130110)~~

摘　　要：目的探讨含有人骨形态发生蛋白-2(hBMP-2)cDNA的真核表达载体在兔骨髓基质干细胞(MSCs)中的转录及表达情况,为进一步进行多基因转染MSCs复合纳米仿生骨治疗骨缺损实验提供材料。方法用电穿孔方法将真核表达载体pIRES2-EGFP-hBMP2转染至兔MSCs中,荧光显微镜及流式细胞学检查观察转染效果、检测转染效率,定量RT-PCR方法观察hBMP-2cDNA在兔MSCs中的转录情况,免疫组织化学及westernblot方法检测hBMP-2蛋白表达情况,ALP活性定量测定检测hBMP-2蛋白功能。结果电穿孔方法将重组质粒转染至兔MSCs中,荧光显微镜下观察到绿色荧光蛋白表达,流式细胞仪检测转染效率为(42.7±2.1)%,转染24h后定量RT-PCR方法检测到hBMP-2cDNA在兔MSCs中的转录片断,免疫组织化学观察到hBMP-2蛋白呈强阳性表达,westernblot方法可见18KDhBMP-2蛋白条带,转染组细胞ALP活性明显提高(P>0.05)。结论pIRES2-EGFP-hBMP2通过电穿孔方法导入兔MSCs,并在其中有效表达,发挥生物学作用。Objective To investigate the transcription and expression of hBMP-2 cDNA via eukaryotic expression vector in rabbit MSCs in vitro so as to provide materials for further experiments on treatment of bone defects by bionic nanobone carried with muhigene-transfected MSCs. Methods The p1RES2-EGFP-hBMP2 plasmids were transfeeted into rabbit MSCs by means of eleetroporation. The efficiency of transfeetion was evaluated by fluorescence microscopy and flow eytometry assay. Quantitative RT-PCR was resorted to for observation of successful transcription of hBMP-2 gene. Immunohistoehemistry and western blot assay were used to identify the protein expression of hBMP-2 gene. The function of hBMP-2 was showed by quantitative analysis of ALP activity. Results Plasmids pIRES2-EGFP-hBMP2 could be transfeeted into rabbit MSCs through eleetroporation method. Green fluorescent protein was visible under fluorescence microscopy and the transfeetion efficiency was （42.7 ± 2. 1 ）% by flow eytometry assay. The hBMP-2 gene fragments were detected by RT-PCR. The protein was observed to be strongly positive in immunohistoehemistry and had 18 KD demonstration in western blot assay. The ALP activity of tranfeeted MSCs was obviously increased（ P 〉 0. 05） . Conclusion The recombinant plasmid containing hBMP-2 cDNA can be transfeeted and expressed effectively in rabbit MSCs.

关 键 词：人骨形态发生蛋白-2 骨髓基质干细胞 基因转染 表达 

分 类 号：R318.0[医药卫生—生物医学工程]