TGF-β1对骨髓间充质干细胞迁移力及Snail表达的影响

Effects of Transforming Growth Factor β1 on the Migration of Bone Marrow Mesenchymal Stem Cells and the Expression of Snail

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摘要采用密度梯度离心结合贴壁法分离、培养大鼠骨髓间充质干细胞(BMMSCs),用免疫荧光法对培养第3代的细胞进行鉴定。用改良的Transwell小室及MTT法检测不同浓度TGF-β1对细胞迁移力及细胞增殖的影响;流式细胞仪检测细胞凋亡;RT-PCR检测TGF-β1作用不同时间细胞snail、基质金属蛋白酶2(MMP-2)mRNA表达;免疫荧光和Western印迹检测snail表达情况。结果表明密度梯度离心结合贴壁法能有效分离、纯化大鼠BMMSCs,免疫荧光检测显示培养的细胞CD29、CD44表达阳性,而CD34、CD45表达阴性;外源性TGF-β1对BMMSCs迁移力的促进作用具有剂量依赖性,在2ng/ml时达到最高。高浓度却抑制BMMSCs的迁移。在2ng/mlTGF-β1刺激下,细胞凋亡明显降低,snail、MMP-2mRNA及snail表达明显增高,但对细胞增殖无明显影响。通过研究TGF-β1对BMMSCs的迁移力的影响及作用机制,为体外调控BMMSCs高效迁移入脑从而发挥其修复神经损伤作用提供实验依据和理论基础。 Density gradient centrifugalization combined with adherence method were used to segregate and cultivate rat bone marrow mesenchymal stem cells （BMMSCs）. BMMSCs cultivated to the 3rd passage were characterized using immunofluorescence technique. The effects of different concentrations of transforming growth factor β1 （TGF-β1） on the migration and proliferation were detected by the modified Transwell chambers and MTT. Cell apoptosis was detected by flow cytometry （FCM）. The effects of TGF-β1 for different time points on the expression of snail and MMP-2 mRNA was measured by RT-PCR, and snail protein expression was detected by immunofluorescence technology and Western blot. Results demonstrated that density gradient centrifugalization combined with adherence method could segregate and purify rat BMMSCs effectively. The results of immunofluo- rescence stain showed that CD29 and CD44 expression were positive while CD34 and CD45 expression was negative for BMMSCs. The TGF-β1 induced a dose-dependent increase in cell migration, which got the peak at 2 ng/ml, and high concentration of TGF-β1decreased the cell migration. Cell apoptosis was reduced with 2 ng/ml TGF-β1 and had obvious difference with control groups （P〈0.05）. The expression level of snail mRNA TGF-β1 treatment at 6 h and the expression level of MMP-2 mRNA at 12 h were significantly higher than the expression level of those at other time points. The protein expression level of snail in TGF-β1-treated group at 24 h was significantly higher than that of control group, but it had no effect on cell proliferation. The study will investigate the possibly molecular mechanisms of TGF-β1on the invasive power of BMMSCs and the expression of snail, and afford experimental foundation for controlling BMMSCs’ migrating into brain effectively in vitro.