1,25(OH)_2D_3对体外人骨髓基质细胞成脂及成骨分化的影响

The effects of 1,25(OH)_2D_3 on adipocytic and osteogenic differentiation of human marrow stromal cells in vitro

目的观察1,25(OH)2D3对骨髓基质细胞(MSCs)的成脂及成骨分化的影响。方法以离心法分离培养人骨髓基质细胞(MSCs),以1,25(OH)2D3作为分化诱导剂对细胞进行干预,采用MTT法测细胞增殖率;测细胞浆碱性磷酸酶(ALP)含量,并用细胞碱性磷酸酶染液试剂盒及苏丹Ⅲ分别对成骨细胞和脂肪细胞进行组织化学染色,计数。结果1,25(OH)2D3干预8天后,实验组的吸光度值低于对照组,二者间有显著差异(P<001)。实验组经1,25(OH)2D3干预12天,胞浆ALP含量高于对照组,二者间的差异有显著性(P<001)。细胞染色结果表明实验组苏丹Ⅲ+细胞百分比较对照组减少,两组间有显著性差异(P<005);ALP+细胞百分比较对照组增加,二者间的差异有显著性(P<001)。结论1,25(OH)2D3对骨髓基质细胞增殖有轻度抑制作用,促进MSCs的成骨分化,抑制其成脂分化,同时上述结果也反映了成骨细胞与脂肪细胞间存在的反变关系,从而进一步表明二者来源于同一前体细胞的可能性。

Objective To observe the effects of 1,25(OH)_2D_3 on adipocytic and osteogenic differentiation of human marrow stromal cells(MSCs) in vitro. Methods The primary human marrow stromal cells(MSCs) were purified from the femur marrow by density gradient centifugation method. In subcultures, human marrow stromal cells(MSCs) were treated with 10^-8 mol/L 1,25(OH)_2D_3 which acted as a differentiation inducer. The proliferation of human MSCs was measured using MTT method; cells alkaline phosphatase (ALP)activity was measured; the histochemical staining of cells was performed with Sudan Ⅲ which was used to identify adipocytic cells and ALP stain which was used to identify osteogenic cells and the numbers of Sudan Ⅲ-positive cells and ALP-positive cells were seperately determined by counting under a light microcope. Results After the cells of the test grope were treated with 1,25(OH)_2D_3 for 8 days,the optical density values were significantly lower than that in the control(P<0.01). Treatment of cells in test group with 1,25(OH)_2D_3 for 12 days led to a significant increase in ALP activity(P<0.01). The percentage of Sudan Ⅲ-positive cells in test group was significantly lower than that in the control(P<0.05) while the percentage of ALP-positive cells was significantly higher than that in control(P<0.01). Conclusion 1,25(OH)_2D_3 can slightly inhabit the proliferation of human MSCs; 1,25(OH)_2D_3 induce human MSCs differentiation into osteoblasts and inhabit adipocytic differentiation. There was an inverse relationship between the differentation of adipocytic and osteogenic cells in human MSCs cultures which is consistent with the possibility that the regulation of adipogenesis and osteogenesis can occur at the level of a common precursor in vivo.