辛伐他汀对BMSCs成骨分化过程中WNT信号通路早期WNT3的影响

Effects of simvastatin on WNT3 of WNT signal pathway in the early process of osteoblast differentiation of bone marrow stromal cells of rats

目的进一步研究辛伐他汀(simvastatin,SIM)体外给药对SD大鼠骨髓基质干细胞在向成骨细胞分化过程中WNT信号通路早期WNT3表达的影响,进一步探讨辛伐他汀在WNT信号通路的作用及其作用机制。方法10只4周龄雌性SD大鼠,颈椎脱位法处死后取股骨骨髓细胞和胫骨骨髓细胞向成骨方向诱导培养。应用培养的第二代细胞随机分为实验组及对照组。实验组加入10^-7mol/L的SIM(先应用培养基稀释SIM溶于DMSO后制成的母液),对照组加入含有相同量的DMSO培养基。两组细胞在加入SIM 12h和36h后分别提取RNA和蛋白质,采用实时定量-PCR法检测WNT3 mRNA的表达,采用Western blot法检测两组细胞WNT3蛋白的表达。结果(1)实时定量-PCR结果:SIM干预12h后WNT3 mRNA的表达水平实验组与对照组相比表达增高,两组间差异有统计学意义(P<0.01)。SIM干预36h后WNT3 mRNA的表达水平实验组与对照组相比表达增高,两组间差异有统计学意义(P<0.05)。(2)Western blot结果:SIM干预12h及36h后WNT3蛋白表达水平实验组与对照组相比无显著增高,两组间差异均无统计学意义(P>0.05)。结论辛伐他汀在促进骨髓基质干细胞成骨细胞分化的早期发挥了作用,这其中涉及到Wnt信号通路的活化,但是在不同的时间点所起作用有所不同。

Objective To research the effects of simvastatin on WNT3 of WNT signal pathway in the early process of osteoblast differentiation of bone marrow stromal sells( BMSCs) of SD rats in vitro,to further study the role of simvastatin in the signal pathway of WNT and to further clarify the mechanism. Methods Ten 4-week-old female Sprague-Dawley rats were killed by cervical dislocation. The bilateral femur and tibia bone marrow cells were obtain under sterile conditions,which were then cultured in vitro. Two groups were established: experimental and control groups. After two passage,all the cells were treated with osteogenic inductive DMEM and experimental group were added 10^-7 mol/L SIM at the same time. Two groups were respectively extracted protein and RNA after 12 h and 36 h. Real-time RT-PCR was performed for evaluating the expression level of WNT3 mRNA.Western Blot was performed for evaluating the expression level of WNT3 protein. Results( 1) The result of real time-PCR: After12 h treatment of SIM: the expression level of mRNA of WNT3 was significantly higher in the experimental group compared to the control group( P<0. 01). After 36 h treatment of SIM: the expression level of mRNA of WNT3 was significantly higher in the experimental group compared to the control group( P<0. 05).( 2) The result of Western Blot: After 12 h and 36 h treatment of SIM:the expression level of WNT3 protein was not significantly higher in the experimental group compared to the control group( P>0. 05).Conclusion There are some effects of simvastatin on osteoblast differentiation of bone marrow stromal sells( BMSCs) of SD rats in vitro. This involves the activation of the WNT signaling pathway,but the role at different time points was different.