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目的观察不同强度的恒磁场作用不同时间对体外培养的大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)向成骨细胞分化的影响及机制。方法密度梯度离心法分离大鼠骨髓单核细胞进行体外培养。以3~4代的MSCs为靶细胞,对其进行诱导分化和施以恒磁场处理。磁场暴露强度分别为0、0.5、1.5、2、3 mT。以IFCC推荐法检测碱性磷酸酶(alkaline phosphatase,ALP)活性表达;茜素红染色观察钙结节的数量和大小;放免法检测骨钙素(osteocalcin,OCN)和Ⅰ型胶原的分泌;Western blot分析MAPK P38信号分子的磷酸化,并探讨其特异性阻断剂对恒磁场调控MSCs成骨分化的影响。结果①恒磁场明显促进Ⅰ型胶原和ALP的活性表达,增加OCN的分泌及钙结节的形成,其中1.5~2 mT磁场强度促MSCs成骨分化作用最为明显;②恒磁场促进磷酸化MAPK P38的表达;③加入P38信号通路SB253580明显抑制恒磁场的促成骨效应。结论磁场促进MSCs成骨分化,其机制与P38信号分子的激活有关。
OBJECTIVE: To observe the effects of different intensities of constant magnetic field on the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into osteoblasts in vitro and its mechanism. Methods Rat bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in vitro. 3 to 4 generations of MSCs as target cells, their induction and differentiation and applied constant magnetic field treatment. Magnetic field exposure intensity were 0,0.5,1.5,2,3 mT. The alkaline phosphatase (ALP) activity was detected by IFCC method. The number and size of calcium nodules were observed by alizarin red staining. The secretion of osteocalcin (OCN) and collagen Ⅰ was detected by radioimmunoassay. blot analysis of phosphorylation of MAPK P38 signaling molecules and explore the effect of specific blockers on osteogenic differentiation of MSCs induced by constant magnetic field. Results ① Constant magnetic field significantly promoted the expression of collagen type Ⅰ and ALP, increased the secretion of OCN and the formation of calcium nodules. The magnetic field intensity of 1.5 ~ 2 mT promoted the osteogenic differentiation of MSCs most obviously; ② The constant magnetic field promoted the phosphorylation of MAPK P38 The expression of P38 signal pathway SB253580 obviously inhibited the effect of constant magnetic field on bone formation. Conclusion Magnetic field can promote the osteogenic differentiation of MSCs, and its mechanism is related to the activation of P38 signaling molecules.