P53特异性抑制剂PFT-α诱导骨髓间充质干细胞向心肌样细胞的分化

背景：最新研究表明,P53可通过阻断P53-P21蛋白通路,显著提高干细胞分化率。而5-氮杂胞苷主要通过激活P53-P21蛋白通路,抑制细胞增殖,导致细胞凋亡。目的：观察P53特异性抑制剂PFT-α对大鼠骨髓间充质干细胞向心肌样细胞分化的影响。方法：分离SD大鼠骨髓间充质干细胞进行培养、传代,取第4代细胞分为4组,正常对照组、PFT-α组、5-氮杂胞苷组及PFT-α＋5-氮杂胞苷组。结果与结论：原代培养的骨髓间充质干细胞传代诱导后细胞体积变大,呈长梭形,排列趋一致。当PFT-α浓度≤20μmol/L时,能减少骨髓间充质干细胞凋亡。心肌样细胞鉴定结果显示,诱导后4周时,可见正常对照组少量表达肌钙蛋白Ⅰ和CX-43,其余3组均强表达。心肌细胞分化率结果显示,诱导4周时,PFT-α组和5-氮杂胞苷＋PFT-α组显著高于5-氮杂胞苷组。Western Blotting检测结果示,诱导1周时,5-氮杂胞苷组P53、P21表达最强,PFT-α组几乎不表达;诱导4周时,5-氮杂胞苷组、PFT-α组、PFT-α＋5-氮杂胞苷组P53、P21表达明显高于正常对照组。PFT-α组、PFT-α＋5-氮杂胞苷组内诱导4周时P53、P21表达量均高于1周时表达。提示通过P53抑制剂PFT-α阻断P53-P21蛋白通路,能显著减少骨髓间充质干细胞凋亡,促进其增殖,且能诱导其向心肌样细胞分化。

Differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells induced by P53 specific inhibitor p-fifty three inhibitor-alpha

BACKGROUND： Latest researches have shown that P53 can significantly increase the differentiation rate of stem cells by locking the protein pathway P53-P21. While 5-azacytidine can inhibit cell proliferation and lead to apoptosis by activating the P53-P21 protein pathway. OBJECTIVE： To investigate the effects of P53 specific inhibitor p-fifty three inhibitor-α on differentiation of rat bone marrow mesenchymal stem cells into cardiomyocyte-like cells.METHODS： Bone marrow mesenchymal stem cells were separated from Sprague-Dawley rats for culturing and passage. Passage 4 bone marrow mesenchymal stem cells were divided into four groups： normal control group, p-fifty three inhibitor-α group, 5-azacytidine and p-fifty three inhibitor-α＋5-azacytidine group. RESULTS AND CONCLUSION： Primary bone marrow mesenchymal stem cells were smaller, and after passaged and induced, the cells were larger than primary cells and exhibited spindle-shaped appearance and arranged in order. MTT assay showed that p-fifty three inhibitor-α at 0-20 μmol/L could reduce the apoptosis of bone marrow mesenchymal stem cells. Identification results of cardiomyocyte-like cells showed that after induced for 4 weeks, little expression of cardiac troponin Ⅰ and CX-43 could be seen in the normal control group, and the expression of cardiac troponin Ⅰ and CX-43 in the other three groups was strong. After induced for 4 weeks, the differentiation rate of cardiomyocyte-like cells in the p-fifty three inhibitor-α and p-fifty three inhibitor-α＋ 5-azacytidine group was significantly higher than that in the 5-azacytidine group. Western blotting analysis showed that at 1 week after induction, the expression of P53 and P21 in 5-azacytidine group was strongest compared with the other groups, but no expression was observed in p-fifty three inhibitor-α group. At 4 weeks after induction, the expression levels of P53 and P21 in 5-azacytidine group, p-fifty three inhibitor-α group and p-fifty three inhibitor-α＋5-azacytidine group were higher than those in the normal control group. The expression levels of P53 and P21 in p-fifty three inhibitor-α group and p-fifty three inhibitor-α＋5-azacytidine group at 4 weeks after induction were higher than those at 1 week after induction. It indicates that P53 specific inhibitor p-fifty three inhibitor-α can significantly induce the apoptosis, promote the proliferation of bone marrow mesenchymal stem cells through blocking P53-P21 protein pathway, and inhibit bone marrow mesenchymal stem cells to differentiate into cardiomyocyte-like cells.