体外诱导成人脂肪间充质干细胞分化为平滑肌细胞的实验研究

Differentiation of adult adipose-derived mesenchymal stem cells into smooth muscle cells induced by PDGFBB in vitro

目的体外培养成人脂肪间充质干细胞(ADMSCs),并应用血小板衍生生长因子-BB(PDGF-BB)诱导ADMSCs分化为平滑肌细胞。方法采用酶消化法和贴壁培养法分离培养ADMSCs,流式细胞仪对第5代细胞进行表面抗原和细胞周期的检测,然后对第5代细胞进行PDGF-BB诱导,于诱导后2周进行免疫组织化学鉴定。结果体外培养的ADMSCs呈梭形,细胞形态均一,传代稳定。干细胞相关标志CD29,CD44表达阳性,内皮细胞相关标志CD31和造血干细胞相关标志CD34表达阴性。ADMSCs中G0/G1,S,G2/M期的细胞分别占90.14%,3.77%,6.09%。定向诱导后倒置显微镜下观察细胞呈长梭状,胞膜清晰,无空泡,可重叠生长,融合后细胞形成"峰"和"谷"状,免疫荧光化学显示诱导组细胞α平滑肌肌动蛋白表达阳性。结论成人脂肪组织中含有间充质干细胞,且可经PDGF-BB诱导分化为平滑肌细胞。

Objective To investigate whether in vitro differentiation of adult adipose-derived mesenchymal stem cells （ADMSCs） into vascular smooth muscle cells can be successful with induction of platelet-derived growth factor BB （PDGF-BB）. Methods ADMSCs were isolated and cultured from adult greater omentum adipose tissue in vitro using enzyme digestion and wall-adhering methods. The morphological characteristics of cells were observed with in- verted microscope. The cell cycle and expression of CD29, CD31, CD34 and CD44 were detected by flow cytometry. The fifth passage cells were induced with PDGF-BB to differentiate into smooth muscle cells. The expression of SMα- actin were detected by immunofluorescence and observed with flurescence microscope. Results The isolated and cultured ADMSCs were evenly fusiformed across various passages. ADMSCs expressed CD29 and CD44 but not CD31 or CD34 as indicated by flow cytometry. Study of cell cycles revealed that there were 90.14% of the cells in G0/G1, 3.77% in S and 6.09% in G2/M. After two weeks＇ induction with 20 ng/ml PDGF-BB, the cells presented with a spindle shape and a ＂hill-and-valley＂ growth pattern. The induced cells expressed characteristic SMα-actin by immunohistochemistry. Conclusion Adult adipose tissue was shown to contain plenty of productive mesenchymal stem cells that can be in vitro isolated, cultured and differentiated into smooth muscle cells.