rAAV2-hTGF-β1转染犬MSCs诱导分化为软骨细胞的研究  

Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1

作　　者：刘斌[1] 蔡道章[1] 戎利民[1] 董健文[1] 曾春[1] 史德海[1] 张非[1] 

机构地区：[1]中山大学附属第三医院骨科,广州510630

出　　处：《中华显微外科杂志》2009年第3期213-216,F0003,共5页Chinese Journal of Microsurgery

基　　金：广东省科技计划项目（2007B031001004,2005B34001003）;中山大学医科青年教师科研启动基金（2007018）

摘　　要：目的探讨rAAV2-hTGF-β1体外转染犬MSCs定向分化为软骨细胞的可行性。方法应用密度梯度离心法及贴壁筛选法分离培养犬MSCs。倒置显微镜及Giemsa染色观察细胞形态．流式细胞技术鉴定其表面标记物。取第三代MSCs，分别以感染复数（MOI）为1×10^5病毒基因数／细胞（v.g／cell）、5×10^5v．g．／cell的rAAV2-hTGF-β1进行转染。培养后定期取各组细胞进行相关检测。Western blot检测hTGF—β1的表达，ELISA法测定hTGF—β1的含量。RT-PCR检测Ⅱ型胶原、Aggrecan mRNA的表达，免疫细胞化学法检测Ⅱ型胶原的表达。结果原代及传代培养的MSCs呈梭形外观，具有较强的增殖能力。细胞表面抗原CD29、CD44、CD105表达阳性，CD34、CD45表达阴性。rAAV2-hTGF-β1转染MSCs后，Western blot法检测到目的蛋白hTGF—β1稳定表达；ELISA法检测转染组MSCs培养上清液中的hTGF-β1表达，且随时间的延长，各组hTGF—β1浓度逐渐增加，MOI5×10^5组表达量明显高于MOI1×10^5组（P〈0．01）；RT—PCR检测到各转染组Ⅱ型胶原、Aggrecan mRNA表达，免疫细胞化学法检测转染组细胞Ⅱ型胶原呈阳性表达，且高MOI值组表达强度明显高于低MOI值组。结论rAAV2-hTGF—β1转染犬MSCs后可定向分化为软骨细胞，作为软骨组织工程的种子细胞是可行的。Objective To investigate the potential application of human transforming growth factorbeta-1 （hTGF-β1） gene mediated by type 2 recombinant adeno-associated virus （rAAV2） vector inducing chondrogenic differentiation of canine mesenchymal stem cells （MSCs） in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifugation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow cytometry was used to detect surface antigens of MSCs. The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 × 10^5 v.g./cell or 5 × 10^5 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggreean were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 10^5 group and MOI of 1 × 10^5 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two （P 〈 0.01 ）. After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.

关 键 词：间充质干细胞 骨髓 腺相关病毒 转化生长因子β1 软骨细胞 组织工程 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]