Lasp1蛋白和铁蛋白在重组人骨形态发生蛋白2介导的小鼠骨髓间充质干细胞成骨过程中的调控机制研究

Regulatory effects of LASP1 and ferritin on rhBMP2-induced osteogenic differentiation of bone mar-row mesenchymal stem cells

目的体外研究验证小鼠骨髓间充质干细胞（MSCs）中Laspl蛋白及铁蛋白在骨形态发生蛋白2（BMP2）介导成骨中的机制。方法从3-18个月龄的C57BL／6J小鼠体内分离、纯化出MSCs，并传代至第3代后，分为3组：实验组的高糖培养基内加入100μg／L的BMP2及成骨诱导液，阳性对照组加入单纯的成骨诱导液，空白对照组不做处理。3组分别诱导7、14、21d，并采用实时荧光定量聚合酶链反应（RT-qPCR）检测4个成骨相关标记基因：Runt相关转录因子2、骨钙素、Osterix蛋白、骨桥素，以确认是否分化成骨。确认成骨后，采用蛋白印迹法检测成骨结果阳性组细胞中的Laspl蛋白及铁蛋白各自的表达浓度。以慢病毒转染基因沉默技术下调小鼠MSCs的Laspl蛋白的基因表达，并重新进行BMP2介导的诱导成骨过程，再进行上述成骨标志基因的RT-qPCR检测。结果RT-qPCR过程显示实验组和阳性对照组显示诱导成骨，且实验组诱导成骨效果更佳。蛋白印迹法检测结果显示仅实验组中的Laspl蛋白明显表达量下调而铁蛋白表达量明显上调。采用慢病毒转染基因沉默技术下调Laspl蛋白表达量后，成骨相关基因表达量明显上调。结论BMP2可以介导小鼠MSCs诱导成骨，且成骨诱导效果极佳。结合前期研究，我们可以确认Laspl蛋白及铁蛋白参与调控BMP2的信号通路，且Laspl蛋白对BMP2介导的诱导成骨呈抑制作用。

Objective To clarify the function of LIM and SH3 domain protein-1 （LASP1） and ferritin in rhBMP2-induced osteogenic differentiation of beagle bone marrow mesenchymal stem cells （BMSCs）. Methods After BMSCs from 3-18-month-old C57BL/6J mice were cultured adherently for 24 hours, they were subjected to osteogenic differentiation for 7, 14 and 21 days in 3 groups. BMP2 （100 μg/L） and osteogenic differentiation medium was added in the experimental group, only osteogenic differentiation medium was added in the control group, and nothing was added in the blank group. Osteoblast differentiation was determined by examining marker genes （Runx2, OSX, OCN and OPN） using qRT-PCR. The protein expression of both LASP1 and ferritin was investigated using western blotting. After LASP1 and ferritin were silenced in the cells in the experimental group after transfection of shRNA to target LASPI（m）, rhBMP2-indueed osteogenesis was repeated to verify the roles of LASP1 and ferritin in osteoblast differenti-ation. Results The qRT-PCR showed successful osteoblast differentiation in the experimental group. Western blotting verified significant down-regulation of LASP1 and up-regulation of ferritin in the experimental group. After the LASPt gene was silenced, the expression levels of osteoblast differentiation marker genes in the experimental group were higher than those in the control group. Conclusions rhBMP2 can induce mouse BMSCs to differentiate into osteoblasts in a significant manner. Combined with our preliminary re- search, the present study may confirm that LASP1 and ferritin, which play an important role in regulating eytoskeleton activity and iron metabolism, are critical in the osteogenic differentiation of mouse BMSCs in- duced by rhBMP2.