ER-α-ERK5信号通路介导流体剪切力促进MC3T3-E1细胞增殖的研究

发布时间：2018-10-09 18:06

【摘要】：背景:随着现代社会生活的改变,骨质疏松症、骨折延迟愈合和骨折不愈合的发病率逐年上升。机械应力在骨的形成、骨的塑形及骨动态平衡的维持中都发挥着重要作用,但这过程离不开雌激素的协同作用及相关信号分子对力学信号的传导。因此,研究和阐明机械应力和雌激素对成骨细胞作用的具体机制对骨质疏松症、骨折延迟愈合和骨折不愈合的预防和治疗有重要意义。流体剪切力是研究最多的一种机械应力模式。本实验室前期研究已表明,流体剪切力可以通过活化ERK5促进成骨细胞的增殖,但其具体分子机制知之甚少。目的:在本实验中对小鼠成骨MC3T3-E1细胞加载12dyne/cm2的流体剪切力,验证流体剪切力通过活化ERK5促进了成骨细胞的增殖,并探索下一步实验中流体剪切力的加载时间;研究雌激素受体α(ER-α)在流体剪切力促进细胞外信号调节激酶5(ERK5)活化中的作用;探索ER-α-ERK5信号通路在成骨细胞增殖中的具体分子机制。方法:给予小鼠成骨MC3T3-E1细胞孵育特异性ER-α抑制剂MPP(methyl-piperidino-pyrazole,1μmol/L),孵育高选择性ERK5活性抑制剂XMD8-92(5μmol/L),和/或加载12 dyn/cm2流体剪切力处理。采用MTT实验检测成骨细胞的增殖活性,采用蛋白免疫印记实验检测ER-α、ERK5、p-ERK5、Cyclin D1(细胞周期蛋白)和CDK4(细胞周期蛋白依赖性激酶)蛋白的表达水平。结果:生理强度(12 dyn/cm2)的流体剪切力作用于成骨MC3T3-E1细胞60min后能显著促进成骨细胞增殖,促进Cyclin D1和CDK4的表达;同时ER-α和p-ERK5的表达显著增加。成骨细胞孵育MPP抑制ER-α后,p-ERK5的表达显著下降,Cyclin D1和CDK4表达也显著降低。孵育XMD8-92抑制ERK5活性后,流体剪切力促进的成骨细胞中Cyclin D1和CDK4表达水平显著下降。结论:ER-α介导流体剪切力对小鼠成骨MC3T3-E1细胞ERK5的活化;流体剪切力通过ER-α-ERK5信号通路上调Cyclin D1和CDK4的表达水平,最终导致成骨MC3T3-E1细胞增殖。
[Abstract]:Background: with the change of modern society, the incidence of osteoporosis, delayed union and nonunion is increasing year by year. Mechanical stress plays an important role in the formation of bone, the formation of bone and the maintenance of dynamic balance of bone. However, this process can not be separated from the synergistic effect of estrogen and the transmission of mechanical signals by related signaling molecules. Therefore, it is important to study and clarify the mechanism of mechanical stress and estrogen action on osteoblasts for the prevention and treatment of osteoporosis, fracture delayed healing and fracture nonunion. Fluid shear force is one of the most studied mechanical stress modes. Previous studies in our laboratory have shown that fluid shear stress can promote the proliferation of osteoblasts by activating ERK5, but its molecular mechanism is poorly understood. Objective: to investigate the loading time of fluid shear force (12dyne/cm2) on mouse osteoblast MC3T3-E1 cells by activating ERK5 to promote the proliferation of osteoblasts. To study the role of estrogen receptor 伪 (ER- 伪) in the activation of extracellular signal-regulated kinase 5 (ERK5) induced by fluid shear stress, and to explore the molecular mechanism of ER- 伪 -ERK5 signaling pathway in osteoblast proliferation. Methods: mouse osteoblast MC3T3-E1 cells were incubated with MPP (methyl-piperidino-pyrazole,1 渭 mol/L), a highly selective inhibitor of ERK5 activity, XMD8-92 (5 渭 mol/L), and / or treated with 12 dyn/cm2 fluid shear stress. The proliferative activity of osteoblasts was detected by MTT assay, and the expression levels of ER- 伪 ERK5 (p-ERK5) cyclin D1 (cyclin D1) and CDK4 (cyclin dependent kinase) protein were detected by protein imprinting assay. Results: the physiological intensity (12 dyn/cm2) fluid shear stress could significantly promote the proliferation of osteoblasts, promote the expression of Cyclin D1 and CDK4, and increase the expression of ER- 伪 and p-ERK5. The expression of p-ERK5 in osteoblasts was significantly decreased after MPP inhibited ER- 伪. The expression of Cyclin D1 and CDK4 was also significantly decreased. After XMD8-92 inhibited ERK5 activity, the expression of Cyclin D1 and CDK4 in osteoblasts stimulated by fluid shear stress decreased significantly. Conclusion fluid shear stress mediates activation of ERK5 in mouse osteoblast MC3T3-E1 cells induced by fluid shear stress, and fluid shear stress up-regulates the expression of Cyclin D1 and CDK4 through ER- 伪 -ERK5 signaling pathway, which leads to the proliferation of osteoblast MC3T3-E1 cells.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68

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