特异性阻抑DKK-1对小鼠脂肪细胞向成骨细胞转分化的影响研究

DKK-1 antibody can promote mouse's adipocytes to transdifferentiate into osteoblasts

[目的]研究特异性阻抑Wnt信号通路抑制因子DKK-1对脂肪细胞向成骨细胞的转分化的影响,以进一步探讨激素性股骨头坏死的发病机制及其防治。[方法]将小鼠前脂肪细胞3T3～L1诱导分化为脂肪细胞,将分化成功的脂肪细胞分为两组:对照组在DMEM(H)培养基培养,实验组在DMEM(H)+DKK-1抗体培养基培养。两组细胞培养3周后分别行油红O染色、ALP染色及茜素红钙结节染色;RT-PCR检测PPARγ-2、LPL、ALP、OCN及Runx-2基因mRNA的转录水平;Western-blot检测PPARγ-2、Runx-2的蛋白表达水平。[结果]与对照组相比,加入DKK-1抗体的实验组的细胞形态呈长梭形,胞浆内未见脂滴沉积,有成骨活性表达,有钙结节形成;RT-PCR结果显示实验组成骨因子Runx-2、ALP及OCN基因mRNA的表达显著高于对照组,而成脂因子PPARγ-2及LPL基因mRNA的表达显著低于对照组(P<0.05);Western-blot检测结果显示实验组PPARγ-2蛋白几乎无表达,而成骨转录分化因子Runx-2蛋白的表达则明显增强,与RT-PCR的结果一致。[结论]在一定条件下,DKK-1抗体可以促使脂肪细胞转分化为具有一定成骨活性的成骨细胞,这为防治激素性股骨头坏死提供了新的思路。

[ Objective] To study whether mouse＇ s adipocytes can be induced to transdifferentiate into osteoblasts by in- hibiting the inhibitor of Wnt signaling pathway, DKK -1, and make further exploration on the pathogenesis and prevention ster- oid - induced osteonecrosis of the femoral head. [ Methods] The mature adipocytes, differentiated from the mouse 3T3 - LI pre -adipocytes, were divided into 2 groups： the control group was cultured in DMEM （H） medium, and the experimental group was cultured in DMEM （H） medium supplemented with DKK - 1 antibody. At the time of third week after intervention, two groups of cells were observed using oil red O staining, ALP staining and alizarin red staining, respectively. The levels of PPAR~/ - 2, LPL, ALP, OCN and Rux - 2 mRNA expression were investigated by real - time PCR. Western - blotting was used to eval- uate the lever of cellular PPARγ- 2 and Runx2 protein. [ Results ] Compared with control group, there were no cytoplasmic lipid droplets in the cells of experimental group, but the cells could express osteogenic activity and form calcium nodules. Furthermore, the RT - PCR results showed that the ossification factors of Runx - 2, ALP and OCN expression of mRNA in the experimental group was significantly higher than that of the control group, while the adipogenic factors of PPARγ - 2 and LPL expression of mRNA was significantly lower than in the control group （ P 〈 0. 05 ）, the Western - blot results showed there was almost no PPARγ -2 protein expression in the experimental group, but the osteogenic factor Runx -2 protein expression was significantly enhanced. [ Conclusion] Under certain conditions, DKK- 1 antibody can promote mouse＇s adipecytes to transdif- ferentiate into osteoblasts with osteogenic activity. And this discovery provides us a new idea for the prevention and treatment of steroid - induced osteonecrosis of the femoral head.