连续细胞培养诱导骨髓间充质干细胞分化

Differentiation of bone marrow mesenchymal stem cells induced by continuous cell culture

目的骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)能够在多种诱导条件下向血管平滑肌细胞(vascular smooth muscle cells,VSMCs)方向分化。文中通过采取连续细胞培养的方式,探讨能否体外诱导BMSCs向VSMCs分化。方法采用全骨髓贴壁的方法将BMSCs从骨髓中提取出,并进行体外扩增,流式及三系分化验证BMSCs。将BMSCs随机分为实验组和对照组,实验组采取连续细胞培养的方法,中途不换液,而对照组采取常规方法培养,每2天换液1次。将BMSCs连续培养5 d后,通过免疫荧光、Western blot、Real-time PCR等方法测定VSMCs的特异性标志物。结果 BMSCs阳性表达CD29、CD90,而阴性表达CD45、CD34、CD49 d,而且经成骨、成脂和成软骨诱导后,茜素红、油红、阿辛蓝染色均呈阳性。通过连续细胞培养5 d后,实验组细胞的α-SMA、Calponin1的蛋白表达水平均较对照组明显增高[(0.84±0.04)vs(0.19±0.02)、(0.87±0.03)vs(0.05±0.06),P<0.01],且免疫荧光实验阳性。实验组较对照组α-SMA mRNA、CNN1 mRNA、SM-MHC mRNA、SM22 mRNA表达升高[(9.33±0.14)vs(1.00±0.18)、(12.64±0.23)vs(1.00±0.17)、(4.75±0.28)vs(1.00±0.23)、(10.26±0.29)vs(1.00±0.22)],差异有统计学意义(P<0.01)。结论在缺乏外源性刺激的情况下,通过采取连续细胞培养的方式,可成功诱导BMSCs向VSMCs分化。

Objective Bone marrow mesenchymal stem cells（ BMSCs） can be induced to the differentiation into vascular smooth muscle cells in many induction conditions. We sought to explore the possibility of the differentiation of mesenchymal stem cells into vascular smooth muscle cells by continuous cell culture in vitro. Methods Rat BMSCs were isolated from the bilateral tibial and femoral bones by the method of whole bone marrow adherence,followed by ex vivo expansion. BMSCs were identified by flow cytometry and three-lineage differentiation. After continuous five days＇ cell culture of BMSCs,the specific surface antigens of VSMCs were detected by immunofluorescence,western blot and real-time PCR. Results BMSCs expressed CD29、90,in contrast,they did not express CD45、34、49d. After induction of osteogenesis,adipogenesis and chondrogenesis,alizarin red、oil red and alcian blue staining produced a strong reaction in cells. The expressions of α-SMA、Calponin1、SM-MHC and SM22 in the cells of experimental group were notably increased,which indicated that BMSCs were differentiating towards VSMCs. Conclusion In the absence of exogenous stimulation,BMSCs can be successfully induced to differentiate into VSMCs by continuous cell culture.