Piezo1蛋白在骨髓间充质干细胞向皮肤成纤维细胞转化的影响

The Effect of Piezo1 Protein on the Transformation of Mesenchymal Stem Cells into Fibroblasts in the Skin

以人的骨髓间充质干细胞为种子,在体位构建体外细胞机械牵张应力模型,探究新型机械敏感性离子通道Piezo1在干细胞向皮肤成纤维细胞转化的作用。采用梯度离心与贴壁筛选相结合的方法,体外培养人的骨髓间充质干细胞,隔代培养后,取生长状态良好的第3代干细胞,进行后面的研究。根据预实验结果,将干细胞分成以下几组:0 h机械牵张应力组、6 h机械牵张应力组、12 h机械牵张应力组和48 h机械牵张应力组,以及Piezo1蛋白的抑制剂Gs MTx4组。将各组细胞种植在Flexcell公司的膜性6孔板中,待融合率在80%左右时,进行体外机械牵张应力的干预。然后采用RT-qPCR、Western-blotting以及激光共聚焦免疫荧光的实验方法检测各组细胞中Piezo1的表达水平,以及干细胞向表皮成纤维细胞的转化水平。原代骨髓间充质干细胞大多呈短梭形,Piezo1表达水平较低。在周期性机械牵张应力的干预下,细胞向成纤维细胞的长梭形的形态学上发展,并且随着时间的延长,变化越明显。RT-qPCR、Western-blotting以及激光共聚焦免疫荧光均发现Piezo1和Vimentin蛋白的表达水平随着干预时间的延长,其表达量也相应增加。机械敏感性离子通道Piezo1蛋白可以介导骨髓间充质干细胞向表皮成纤维细胞转化,为体外构建组织工程皮肤提供种子细胞。

Using human bone marrow mesenchymal stem cells as seeds, an in vitro cell mechanical stretch stress model was constructed in order to explore the role of the new mechanically sensitive ion channel Piezo1 in the transformation of stem cells into skin fibroblasts. Human bone marrow mesenchymal stem cells were cultured in vitro by gradient centrifugation and adherent screening, After subculture, take third-generation stem cell growth in good condition, on the back of the research. According to the preexperimental results, the stem cells were divided into the following groups: 0 h mechanical stretch stress group, 6 h mechanical stretch stress group, 12 h mechanical stretch stress group and 48 h mechanical stretch stress group, and the Piezo1 inhibitor Gs MTx4 group. The cells of each group were planted in the membrane 6 orifice plate of Flexcell, and when the fusion rate reached about 80%, the intervention of external mechanical stretch stress was performed. Then, the expression levels of Piezo1 in each group of cells were detected by RT-qPCR, Wester-blotting and laser co-focusing immunofluorescence, and the transformation level of stem cells into fibroblasts from the epidermis. The primary bone marrow mesenchymal stem cells were mostly in short spindle shape, and the expression level of Piezo1 was low. Under the intervention of cyclic mechanical stretch stress, the morphological development of the long fusiform of cells into fibroblasts was developed, and the change was more obvious with the extension of time. RT-qPCR, Westernblotting and laser co-focusing immunofluorescence found that the expression level of Piezo1 was increased with the extension of intervention time. Piezo1 protein can mediate the transformation of mesenchymal stem cells into fibroblasts and provide seed cells for tissue engineering in vitro.