凝血酶促进骨髓间充质干细胞增殖的作用与机制

Promoting Effect of Thrombin on Proliferation of Bone Marrow-derived Mesenchymal Stem Cells and Its Mechanisms

本研究旨在探讨凝血酶促进人间充质干细胞(MSC)增殖及其机制。在无血清基础培养液中添加不同浓度凝血酶,用MTT实验检测人骨髓MSC增殖状态,PCR检测细胞蛋白酶活化受体(PAR)和c-MYC表达,Western blot分析信号通路的变化,并通过特异性抑制剂阻断凝血酶受体和信号通路,验证凝血酶作用的机制。结果显示,凝血酶可显著刺激MSC增殖,效应呈浓度依赖性;当凝血酶浓度为0.5 U/ml时,细胞增殖明显提高,浓度为8 U/ml时,刺激活性达峰值(P<0.01);PCR结果表明,MSC表达PAR-1和PAR2;当PAR1受体被阻断后凝血酶的促增殖效应被抑制,而PAR2受体被阻断后凝血酶对增殖结果影响不明显;凝血酶刺激MSC后,信号分子AKT发生磷酸化,c-MYC基因表达上调;当AKT信号通路被抑制后,c-MYC表达被抑制,凝血酶刺激细胞增殖效应被明显抑制。结论:凝血酶可通过PAR1受体介导的AKT信号通路的活化,上调MYC基因的表达,从而促进间充质干细胞的增殖。

This study was aimed to investigate the growth-promoting activity of thrombin on mesenchymai stem cells （MSC） and its mechanisms. Human bone marrow MSC were cultured in serum-free medium supplemented with graded concentrations of thrombin, and the proliferation status of MSC was detected by MTT test. The expression levels of protease-activated receptors （PAR） and c-MYC gene were detected by PCR. Activated Akt signaling pathway was revealed by Western blot, and specific inhibitors of the signaling pathways were used to confirm the effects. The results showed that thrombin stimulated MSC proliferation in a dose-dependent manner; the minimal concentration of thrombin for stimulating MSC growth was 0.5 U/ml, and the promoting effect reached its maximum when thrombin at a dose of 8 U/ml was employed. PCR results showed that MSC expressed the two types of PAR1 and PAR2. After PAR1 was blocked with a specific inhibitor SCH79797, the growth-promoting effect of thrombin was inhibited, while this pheno- menon was not observed when MSC were exposed to FSLLRY-NH2, a specific inhibitor for PAR2. Further experiments showed that after exposure to thrombin, the AKT signaling pathway in MSC was promptly activated, and c-MYC expression was greatly up-regulated. Meanwhile, when LY294002, a specific AKT inhibitor, was added into the culture medium, the up-regulation of c-MYC expression was reduced, accompanied by the low rate of MSC growth. It is concluded that thrombin can stimulate MSC proliferation by eliciting PARl-mediated AKT activation and subsequent up- regulation of c-MYC expression.