VEGF165、BMP2双基因转染SD大鼠BMSCs的实验研究  

Experimental study on VEGF165 and BMP2 double genes transfecting into BMSCs in SD rats

作　　者：温洋 陈丽媛 沈师 卓乃强 WEN Yang;CHEN Liyuan;SHEN Shi;ZHUO Naiqiang(First Department of Orthopedic Centre,Suining Municipal Central Hospital,Suining,Sichuan 629000,China;Department of Orthopedics and Joint Surgery,Affiliated Hospital of Southwest Medical University,Luzhou,Sichuan 646000,China)

机构地区：[1]四川省遂宁市中心医院骨科中心一病区,629000 [2]西南医科大学附属医院骨与关节外科,四川泸州646000

出　　处：《重庆医学》2020年第20期3331-3336,共6页Chongqing medicine

基　　金：四川省科技厅-泸州市科技局-泸州医学院联合科研课题(14JC01653-LH75)。

摘　　要：目的探究血管内皮生长因子165(VEGF165)、骨形态发生蛋白(BMP2)双基因转染SD大鼠骨髓基质干细胞(BMSCs)及其基因表达与体外成骨能力。方法提取SD大鼠胫骨骨髓,原代培养获得BMSCs并进行流式细胞学鉴定。通过脂质体(Lipofect-amine2000)介导VEGF165、BMP2基因转染SD大鼠BMSCs,并分为pIRES-hBMP2-hVEGF165转染组(双基因转染)、pIRES-hBMP2转染组(单基因转染)、pIRES-hVEGF165转染组(单基因转染)、空载转染组(空白对照)。采用四甲基偶氮唑盐微量酶反应比色法(MTT)、酶联免疫吸附试验、组织学染色方法评估基因转染BMSCs的增殖情况、基因表达及体外成骨能力。结果流式细胞鉴定培养的BMSCs表面Marker示CD44和CD90标志物阳性,CD34和CD45标志物为阴性,证实成功获取大鼠BMSCs。通过荧光显微镜观察各转染组BMSCs,24 h后可见绿色荧光发出,表明转染成功。MTT结果显示:随着时间的延长,各转染组BMSCs细胞的数量呈上升趋势,与未转染BMSCs的相比,细胞数量峰值无明显差异,证实转染后BMSCs生长情况良好。ELISA染色结果显示:pIRES-hBMP2-hVEGF165转染组BMP2蛋白及VEGF165蛋白水平较pIRES-hBMP2转染组和pIRES-hVEGF165转染组及空载转染组显著增高,差异有统计学意义(P<0.05)。组织学染色观察发现pIRES-hBMP2-hVEGF165转染组细胞碱性磷酸酶、骨胶原蛋白及骨钙素阳性率明显高于pIRES-hBMP2转染组、pIRES-hVEGF165转染组、空载转染组,差异有统计学意义(P<0.05)。结论双基因转染后的BMSCs目的基因的表达良好,BMP2、VEGF165基因表达水平较单基因转染明显提高;双基因共转染SD大鼠BMSCs能够获得比单基因转染更优良的体外成骨能力。Objective To investigate the vascular endothelial growth factor 165(VEGF165)and bone morphogenetic protein 2(BMP2)double genes transfecting into the bone marrow stromal stem cells(BMSCs)of SD rats and their gene expressions and in vitro osteogenic capability.Methods The SD rat tibia bone marrow was extracted,BMSCs were obtained via the primitive culture.The cytometry cytological identification was performed.The VEGF165 and BMP2 genes were mediated to transfect into BMSCs of SD rats via lipidosome(lipofect-amine2000)and BMSCs were divided into the pIRES-hBMP2-hVEGF165 transfection group(double genes tranfection);pIRES-hBMP2 transfection group(single gene transfection);pIRES-hVEGF165 transfection group(single gene transfection)and empty loading transfetion group(blank control).The proliferation,gene expression and in vitro osteogenic ability of genes transfected BMSCs were evaluated by adopting MMT,ELISA and histological staining methods.Results The flow cytometry identified that the surface Marker of cultured BMSCs showed the CD44 and CD 90 markers positive,CD34 and CD 45 markers negative,which verified that the rat BMSCs were successfully obtained.The green fluorescence was emitted in each transfection groups after 24 h by fluorescence microscopic obserrvation,indicating successful transfection.The MTT results indicated that the number of BMSCs in the transfected groups showed the increasing trend with the time prolonging,and there was no significant difference in the peak number of BMSCs in the transfection groups compared with the untransfection BMSCs groups,which confirmed that the growth of BMSCs after transfection was good.The ELISA staining results showed that the content of BMP2 protein and VEGF165 protein in the pIRES-hBMP2-hVEGF165 transfection group was significantly increased compared with the pIRES-hBMP2 transfection group,pIRES-hVEGF165 transfection group and empty loading transfetion group,and the difference was statistically significant(P<0.05).The histological staining observation found that the p

关 键 词：骨形态发生蛋白 血管内皮生长因子 基因转染 骨髓基质干细胞 

分 类 号：R336[医药卫生—人体生理学]