骨形态蛋白2诱导大鼠骨髓间充质干细胞分化为心肌样细胞的实验研究

Bone morphogenetic protein 2 induce the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells

目的观察骨形态蛋白2体外诱导骨髓间充质干细胞(BMMSCs)定向分化为心肌样细胞的作用。方法采用密度梯度离心法分离大鼠BMMSCs,取第4代BMMSCs进行诱导:骨形态蛋白2组(BMP-2,终浓度为10μg/L),空白对照组。诱导24h后更换常规培养液继续培养4周。倒置相差显微镜观察细胞的形态学变化,免疫荧光染色法鉴定诱导后BMMSCs中心肌特异性肌钙蛋白I(cTnI)的表达,WesternBlot检测诱导2周和4周后Cx43以及cTnI的表达量,流式细胞计数法计算心肌样细胞诱导分化率。结果原代培养的BMMSCs2周形成集落,呈梭形或星形。传代细胞体积变大,诱导后细胞呈长梭形,呈一致性生长,并出现肌岛样结构。免疫荧光染色结果显示诱导后BMMSCs表达cTnI。WesternBlot检测结果提示诱导后Cx43以及cTnI均明显增多,4周组明显高于2周组。流式细胞计数法显示:BMP-2组心肌样细胞诱导率为(17.9±0.8)%。结论骨形态蛋白2可在体外诱导大鼠BMMSCs定向分化为心肌样细胞,其诱导分化率高。

Objective To observe the effect of the differentiation of bone marrow mesenchymal stem cells （BMMSCs） into cardiomyocyte-like cells with bone morphogenetic protein 2 （BMP-2）. Methods BMMSCs were isolated from bone marrow of SD rats by density gradient centrifugation. The forth passage cells were divided into two groups：BMP-2 group（ 10 p^g/L）, untreated group as control. After 24 h induction, the medium was changed to complete culture medium without any inductor and the cells were culture for 4 weeks. The morphological changes were observed under phase contrast microscope. The cardiomyogenic cells were identified by immanofluorescence staining. The differentiation ratio was examined by flow cytometer. Results BMMSCs of primary culture formed cell colonies at 2 weeks. The passaged cells were larger than those of primary culture. After induction, the cells presented long spindle, aligned in parallel and formed＂ muscle island＂-like structure. The expression of specific protein of cardiac troponin I（cTnI） in induced BMMSCs was positive. Western blot showed that the expression of cTnI and Cx43 in induced BMMSCs was positive. Flow cytometer showed that the differentiation ratio in BMP-2 group were（ 17.9 ± 0. 8）%. Conclusions BMP-2 can induce the differentiation of BMMSCs into cardiomyocyte-like cells, and the differentiation ratio is high.