人转化生长因子β_2基因转染诱导脂肪间充质干细胞向软骨细胞定向分化的实验研究

目的:探讨脂肪间充质干细胞作为软骨组织工程的种子细胞和基因增强的组织工程的可行性。方法:取3周龄Lewis大鼠的腹股沟脂肪垫,消化法获得脂肪间充质干细胞,通过脂质体介导,将hTGFβ2基因转染到体外培养的脂肪间充质干细胞中,采用免疫组织化学染色、RT-PCR和Western blotting的方法检测目的基因与软骨特异性蛋白——Ⅱ型胶原和蛋白多糖表达的情况。结果:从成体大鼠脂肪组织中培养出脂肪间充质干细胞,体外培养呈成纤维细胞样,能大量稳定增殖传代。原代脂肪间充质干细胞能自发分化为脂肪细胞,传代细胞在胰岛素和地塞米松的作用下生成脂滴向脂肪细胞分化;hTGFβ2基因转入脂肪间充质干细胞能获得瞬时及稳定表达,并促使Ⅱ型胶原和蛋白多糖合成。结论:从脂肪组织中可获得具有多分化潜能的间充质干细胞,并能在体外稳定增殖传代,可自发或经诱导后分化为脂肪细胞;pcDNA3.1(+)/hTGFβ2真核表达载体成功转染脂肪间充质干细胞,诱导其向软骨细胞分化。

Study on Chondrogenic Differentiation of Adipose Mesenchymal Stem Cells Transfected with Human Transforming Growth Factor beta-2 Gene in vitro

Objective To investigate the basic biological characteristics and the effect of hTGFβ2 gene transfection on the differentiation of adipose mesenchymal stem cells into cartilage,and to discuss the feasibility on adipose mesenchymal stem cell as seeding cell of cartilage tissue engineering.Methods Adipose mesenchymal stem cells were obtained from inguinal fat pads of Lewis rat with digestion.Adipose mesenchymal stem cells were induced into chondrocytes by hTGFβ2 gene transfection,and then the differentiations were examined with immunohistochemistry,RT-PCR and Western blotting.Results A population of adipose mesenchymal stem cells were successfully isolated from rat adipose tissue.They appeared fibroblast-likely and could stably proliferate and be passaged in vitro.Primary cells could differentiated into adipocytes spontaneously,and passaging cells must be induced to differentiated into adipocytes by insulin and dexamethasone.After transfected with pcDNA3.1(+)/hTGFβ2,the differentiation of adipose mesenchymal stem cells towards chondrocytes was verified by the positive staining of type Ⅱ collagen and aggrecan immunohistochemistry,RT-PCR and Western blotting respectively.Conclusions Mesenchymal stem cells with multilineage potential obtained from rat adipose tissue can differentiated to adipocytes,and can be induced to into chondrocytes through by hTGFβ2 gene transfection.