腺病毒介导人转化生长因子β2基因转染诱导骨髓间充质干细胞向软骨细胞的定向分化

Adenoviral-mediated transfer of human transforming growth factor-beta 2 gene induces directional differentiation of bone marrow mesenchymal stem cells into chondrocytes

背景:前人的工作已证实转化生长因子β1能促进骨髓间充质细胞的增殖,诱导其向软骨细胞分化。目的:进一步验证人转化生长因子β2(human transforming growth factor-β2,hTGFβ2)基因转染诱导骨髓间充质干细胞向成骨细胞定向分化作用。方法:取清洁级3周龄雄性Lewis大鼠16只,用于分离培养骨髓间充质干细胞。通过腺病毒重组载体Ad-hTGFβ2将外源性hTGFβ2基因转染到体外培养的第2代大鼠骨髓间充质干细胞中,48h后采用免疫组织化学染色、RT-PCR和Western blotting的方法检测转染细胞中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。结果与结论:从成体大鼠骨髓组织中培养出骨髓间充质干细胞,体外培养呈成纤维细胞样,能大量稳定增殖传代。Ad-hTGFβ2转染骨髓间充质干细胞获得稳定表达,免疫组织化学染色和Western blotting检测到基因转染细胞中Ⅱ型胶原蛋白的合成表达,RT-PCR检测到Ⅱ型胶原和蛋白多糖aggrecan mRNA的表达。结果提示,从骨髓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;Ad-hTGFβ2成功转染骨髓间充质干细胞,诱导其向软骨细胞分化。

BACKGROUND：Previous studies have shown that transforming growth factor-β1（TGF-β1） contributes to proliferation of bone marrow mesenchymal stem cells（BMSCs）,and induces the differentiation of BMSCs into chondrocytes.OBJECTIVE：To investigate the effect of human TGFβ2（hTGFβ2） transfection on the directional differentiation of BMSCs into osteoblasts.METHODS：A total of 16 clean male Lewis rats aged 3 weeks were selected for isolation of BMSCs.hTGFβ2 gene was transfected into the second passage of rat BMSCs cultured in vitro through Ad-hTGFβ2.48 hours later,immunohistochemical staining,reverse transcription-polymerase chain reaction and Western blotting assay were used to determine target gene and cartilage specific protein-type Ⅱ collagen and aggrecan in transfected cells.RESULTS AND CONCLUSION：A population of BMSCs was successfully isolated from adult rats.They appeared fibroblast-likely and could stably proliferate and subculture in vitro.After transfected with Ad-hTGFβ2,the differentiation of bone marrow mesenchymal stem cells towards chondrocytes was verified by the positive results of type Ⅱ collagen protein expression through immunohistochemistry and Western blotting respectively.RT-PCR detected type Ⅱ collagen and aggrecan mRNA expression.Results suggest that MSCs from bone marrow presented multi-differentiational potential,and BMSCs can proliferate and subculture stably in vitro.Ad-hTGFβ2 is successfully transfected into BMSCs,and induces the differentiation of BMSCs into chondrocytes.