腺病毒介导的CTLA-4Ig基因转染人骨髓间充质干细胞成骨特性研究  

Osteogenic characteristics of hMSCs transfected with adenovirus-mediated CTLA-4Ig gene

作　　者：石东文[1] 代飞[2] 陈希炜[1] 贺伟峰[1] 胡晓红[1] 罗高兴[1] 吴军[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所创伤,烧伤与复合伤国家重点实验室疾病蛋白质组学重庆市市级重点实验室,重庆400038 [2]第三军医大学西南医院骨科,全军矫形外科中心,重庆400038

出　　处：《第三军医大学学报》2007年第9期810-813,共4页Journal of Third Military Medical University

基　　金：国家自然科学青年基金(30300090);疾病蛋白质组学重庆市市级重点实验室专项基金(2003)~~

摘　　要：目的通过腺病毒介导CTLA-4Ig基因转染人骨髓间充质干细胞(human marrowmesenchymal stem cells,hM-SCs),观察被修饰细胞CTLA-4Ig蛋白表达情况,以及基因修饰对hMSCs诱导成骨特性的影响。方法采用荧光显微镜观察腺病毒介导CTLA-4Ig基因转染hMSCs的绿色荧光蛋白表达,流式细胞术检测确定转染率,观察对比转染前后细胞形态和细胞周期,用免疫细胞化学方法检测转染hMSCs的CTLA-4Ig蛋白表达,转染hMSCs成骨诱导培养3周后进行成骨特异性标记检测。结果分离培养的hMSCs CD105表达阳性,CD34表达阴性。重组腺病毒介导的CTLA-4Ig基因转染hM-SCs的转染率为81.14%,CTLA-4Ig基因转染hMSCs(CTLA-4Ig-hMSCs)的形态无明显变化,生长良好。免疫细胞化学结果显示,CTLA-4Ig-hMSCs的CTLA-4Ig蛋白表达阳性,诱导培养3周的CTLA-4Ig-hMSCs,碱性磷酸酶染色呈强阳性,免疫细胞化学检测骨钙素表达阳性,钙的四环素荧光标记法显示细胞间有钙的沉积。结论腺病毒介导的CTLA-4Ig基因转染hMSCs能够分泌CTLA-4Ig蛋白,并保持了成骨分化潜能,可用于异基因hMSCs作为骨组织工程种子细胞的应用研究。Objective To observe CTLA-4Ig protein expression in human marrow mesenchymal stem cells （hMSCs） transfected with adenovirus-mediated CTLA-4Ig gene and the effect of gene modification on hMSCs induced osteogenesis. Methods hMSCs were transfected with adenovirus-mediated CTLA-4Ig gene. The transfection efficiency was identified by observing GFP expression and using FACS detection. The cell shape and cell cycle was compared before and after transfection. CTLA-4Ig protein expression in transfected hMSCs was detected by immunocytochemical method. After induction culture for 3 weeks, the specific markers for osteogenesis were examined. Results The isolated and cultured hMSCs were positive for CD105 and negative for CD34. The transfection efficiency of recombinant adenovirus-mediated CTLA-4Ig gene transfection was 81.14%. hMSCs with CTLA-4Ig gene （CTLA-4Ig-hMSCs） did not show marked changes in cell shape and growth. Immunocytochemical detection showed positive CTLA-4Ig expression in CTLA-4Ig-hMSCs and strongly positive alkali phosphatase staining, as well as positive expression of osteocalcin. Macrocyclin fluorescent labeling indicated there were calcium deposits among hMSCs. Conclusion hMSCs modified with CTLA4Ig gene can secrete target protein CTLA4Ig and maintain the characteristics of osteogenesis, which has the potential for repairing bonedefect in the bone tissue engineering.

关 键 词：HMSCS CTLA-4IG 基因转染 诱导成骨 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R373[医药卫生—基础医学]