细胞间接触诱导大鼠骨髓间质干细胞分化为平滑肌细胞

Cell-to-cell contact induced rat mesenchymal stem cells into smooth muscle cells

目的体外诱导骨髓间质干细胞分化为平滑肌细胞,探讨已分化成熟的平滑肌细胞及细胞因子对骨髓间质干细胞分化的影响。方法分别用骨髓间质干细胞加平滑肌细胞条件培养液;骨髓间质干细胞与平滑肌细胞分层培养;骨髓间质干细胞EGFP标记后,与平滑肌细胞混合培养。用平滑肌细胞抗体SM-a-Actin、Calponin免疫荧光染色,检测骨髓间质干细胞的分化情况。结果骨髓间质干细胞与平滑肌细胞混合培养7d后免疫荧光染色,可见EGFP(绿色)和抗SM-a-Actin、Calponin(红色)的双标细胞存在。而加平滑肌细胞条件培养液与分层培养组,骨髓间质干细胞均不表达Calponin。结论细胞间直接接触对诱导骨髓间质干细胞定向分化为平滑肌细胞起决定作用。

Objective To induce MSCs into SMCs in virto, and investigate whether MSCs differentiation is influenced by the differentiated SMCs or cell factors. Methods MSCs and SMCs were cultured and identified, respectively. MSCs were transfected with pEGFP-N3 by lipofectamine 2000. After 24 hours, the EGFP-labeled MSCs were co-cultivated with SMCs for 7 days. Indirect co - culture of MSCs with SMCs were established using a semi-permeable membrane. And MSCs were cultured with conditioned medium from SMCs.As a control, MSCs were cultured alone for 10 days- 14 days Immunofluorescence analysis was performed by using monoclonal antibodies against smooth muscle α-actin, calponin. Results MSCs co-cultured were stained with antibodies against smooth muscle calponin, SM-a-actin and were visualized using confocal laser scanning microscopy with a 200x objective. Several double-positive cells were detected with direct co-culturing two cell types. However, no cells positive for calponin were detected when MSCs were cultured with SMCs-conditioned medium or indirect co- cultured with SMCs. Conclusion Cell-to-cell contact is exsential for MSCs differentiation to SMCs.