达沙替尼对人骨髓间充质干细胞生物学特性的影响

Effect of dasatinib on biological properties of human bone marrow mesenchymal stem cells

目的:在体外探索达沙替尼对人骨髓来源间充质干细胞(hBMSCs)的活力、迁移、细胞周期和凋亡的影响以及潜在的信号通路,以评估达沙替尼在临床应用中对骨髓造血微环境的影响。方法:CCK-8法检测细胞活力;划痕实验检测细胞迁移;流式细胞术检测细胞周期和凋亡;同时采用吖啶橙/溴化乙啶法检测细胞凋亡;酶联免疫吸附实验检测细胞转化生长因子β1(TGF-β1)和肿瘤坏死因子α(TNF-α)的分泌情况;Western blot检测蛋白激酶B(Akt)蛋白的表达和磷酸化以及cleaved caspase-3的蛋白水平。结果:与对照组相比,达沙替尼(1~10nmol/L)抑制hBMSCs的活力和迁移;在随后的实验中使用的浓度为7 nmol/L。达沙替尼促进细胞凋亡,并使更多细胞的周期阻滞在G_1期。此外,hBMSCs TGF-β1和TNF-α的分泌量显著增加。7 nmol/L达沙替尼组cleaved caspase-3的蛋白水平增加,胞内Akt的蛋白量下调且其磷酸化受到抑制。结论:达沙替尼以浓度依赖性的方式抑制hBMSCs的活力和迁移,并促进TGF-β1和TNF-α的分泌,诱导细胞G_1期阻滞和凋亡;达沙替尼可能通过影响胞内Akt蛋白的表达和磷酸化调控上述细胞学行为。

AIM: To explore the effect of dasatinib on the viability,migration,cell cycle and apoptosis of human bone marrow mesenchymal stem cells( hBMSCs),as well as the underlying signal pathway to evaluate the influence of dasatinib on hematopoietic microenvironment clinically. METHODS: The cell viability was measured by CCK-8 assay.The migration ability was detected by wound healing assay. The cell cycle and apoptosis were analyzed by flow cytometry.Acridine orange/ethidium bromide staining was also used to detected apoptosis. The secretion of transforming growth factor-β1( TGF-β1) and tumor necrosis factor-α( TNF-α) were measured by ELISA. The protein levels of cleaved caspase-3,protein kinase B( Akt) and phosphorylated Akt were determined by Western blot. RESULTS: Compared with control group,dasatinib at 1 ~ 10 nmol/L suppressed the viability and migration ability of hBMSCs,and dasatinib at concentration of 7 nmol/L was adopted in the following assays. Dasatinib promoted apoptosis,and blocked the cell cycle in G_1 phase. In addition,the secretion of TGF-β1 and TNF-α was increased markedly. The protein levels of cleaved caspase-3 was increased,but the protein levels of Akt and phosphorylated Akt were decreased. CONCLUSION: Dasatinib inhibits the viability and migration ability of hBMSCs in a dose-dependent manner,promotes the secretion TGF-β1 and TNF-α,and induces cell cycle arrest and apoptosis. Dasatinib might regulate the biological behaviors of hBMSCs observed above by modulating the expression and phosphorylation of Akt.