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目的探讨他克莫司(FK506)对人骨髓间质干细胞(hBMSCs)增殖及向成骨细胞分化的影响。方法 FK506 0.001~5μmo.lL-1处理hBMSCs细胞中,雌二醇0.01μmol·L-1或咖啡因100μmol·L-1为阳性对照组,作用24 h后用BrdU掺入法检测细胞增殖,在促成骨细胞分化液中作用8 d后用比色法检测碱性磷酸酶(ALP)活性,作用12 d后用邻甲酚酞络合法检测钙沉积量;通过检测磷酸盐释放量间接反映钙调神经磷酸酶(CaN)活性,Western印迹法检测核心结合因子α1亚基(Cbfα1)表达。结果与DMSO对照组相比,FK506 0.001~0.01μmol.L-1促进细胞增殖,但对ALP活性及钙沉积量无影响;FK506 0.5~5μmo·lL-1则呈浓度依赖性地抑制细胞增殖,显著抑制ALP活性及减少钙沉积量(P<0.05)。此外,FK5060.1~5μmo·lL-1呈浓度依赖性地降低CaN活性,与相同浓度FK506呈浓度依赖性地下调Cbfα1的表达效应相一致。结论高浓度FK506可通过CaN/Cbfα1通路抑制hBMSCs增殖及向成骨细胞成骨分化。
Objective To investigate the effects of FK506 on the proliferation and differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into osteoblasts. Methods FK506 0.001 ~ 5μmo.lL-1 cells were treated with 0.01μmol·L-1 estradiol or 100μmol·L-1 caffeine for 24 h, and the proliferation of hBMSCs was detected by BrdU incorporation Induced alkaline phosphatase (ALP) activity 8 days after the differentiation of osteoblasts, the calcium deposition was detected by o-cresolphthalein complexation after 12 days, and the calcium release was indirectly detected by measuring the phosphate release Neuro-phosphatase (CaN) activity and Cbfα1 expression were detected by Western blotting. Results Compared with DMSO control group, FK506 0.001 ~ 0.01μmol.L-1 promoted cell proliferation, but had no effect on ALP activity and calcium deposition; FK506 0.5 ~ 5μmo · L-1 inhibited cell proliferation in a concentration- Significantly inhibited ALP activity and reduced calcium deposition (P <0.05). In addition, FK5060.1 ~ 5μmo · lL-1 decreased CaN activity in a concentration-dependent manner, which was consistent with the down-regulation of the expression of Cbfα1 in the same concentration of FK506. Conclusion High concentrations of FK506 can inhibit proliferation and osteogenic differentiation of hBMSCs through CaN / Cbfα1 pathway.