ERK5信号通路及流体剪切力在骨髓间充质干细胞成骨分化过程中作用的研究

发布时间：2018-01-11 12:07

本文关键词：ERK5信号通路及流体剪切力在骨髓间充质干细胞成骨分化过程中作用的研究　出处：《兰州大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:探讨流体剪切力对骨髓间充质干细胞成骨分化的影响及ERK5信号通路在该过程中所起的作用。方法:用全骨髓贴壁细胞培养法提取昆明小鼠的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),根据其表面标志特点用流式细胞术进行鉴定。设计翘板摇床流体剪切力(fluid shear stress,FSS)加载模型,用ANSYS-CFX软件计算FSS的大小。验证ERK5特异性抑制剂XMD8-92对ERK5磷酸化的抑制作用,并探讨最佳FSS加载时间。按不同干预措施分为8组:对照组、XMD8-92组、成骨诱导液组、成骨诱导液+XMD8-92组、FSS组、FSS+XMD8-92组、FSS+成骨诱导液组、FSS+成骨诱导液+XMD8-92组。培养14天后,进行碱性磷酸酶(ALP)活性测定及茜素红染色,并提取胞内蛋白,用蛋白质印记法分别检测不同分组ERK5、p-ERK5、ERK1/2、p-ERK1/2、Runx-2、Col-1表达量的变化。结果:从骨髓中提取出的细胞为梭形,形态均一,生长迅速,CD29+、CD44+、CD90+、CD34-、CD45-,且能向成骨细胞分化,是骨髓间充质干细胞。在BMSCs中,5μmol/L的XMD8-92作用1小时能很好地抑制ERK5的磷酸化,12 dyn/cm2的FSS加载1小时能很好地激活ERK5磷酸化。成骨诱导液和FSS均能激活ERK1/2的磷酸化(P0.001)。FSS组的成骨分化指标:ALP活性、钙结节含量、Runx-2、Col-1的表达水平均高于对照组(P0.01)。成骨诱导液+XMD8-92、FSS+XMD8-92、FSS+成骨诱导液+XMD8-92三组,以上成骨分化指标也均比没有XMD8-92的单纯成骨诱导液、FSS、FSS+成骨诱导液组高(P0.05)。FSS+成骨诱导液组的各成骨分化指标均比单纯成骨诱导液或FSS组高(P0.05)。结论:FSS能够通过刺激ERK1/2的磷酸化促进BMSCs的成骨分化,且与成骨诱导液具有协同促进作用。ERK5特异性抑制剂XMD8-92可以促进BMSCs的成骨分化,ERK5的磷酸化对BMSCs的成骨分化起一定的抑制作用。
[Abstract]:Objective: to investigate the effect of fluid shear stress on osteogenic differentiation of bone marrow mesenchymal stem cells and the role of ERK5 signaling pathway in the process. Extraction of bone marrow mesenchymal stem cells from Kunming mice by whole bone marrow adherent cell culture. Bone marrow mesenchymal stem cells. BMSCs was identified by flow cytometry according to its surface marker characteristics. The fluid shear stress shear stress FSS loading model was designed. The size of FSS was calculated by ANSYS-CFX software, and the inhibitory effect of XMD8-92, a specific inhibitor of ERK5, on the phosphorylation of ERK5 was tested. The optimal loading time of FSS was discussed and divided into 8 groups according to different intervention measures: control group (XMD8-92), osteoblastic inducer group (XMD8-92), osteoblastic induction fluid (XMD8-92) group. FSS XMD8-92 group and XMD8-92 group were cultured for 14 days. Alkaline phosphatase (ALP) activity and alizarin red staining were performed. Intracellular proteins were extracted and ERK5 p-ERK5 ERK1 / 2 was detected by protein imprinting method. Results: the cells extracted from bone marrow were fusiform, homogeneous, and grew rapidly. CD90 CD34-CD45, which can differentiate into osteoblasts, is a bone marrow mesenchymal stem cell in BMSCs. The phosphorylation of ERK5 was inhibited by 5 渭 mol/L XMD8-92 for 1 hour. 12 dyn/cm2 FSS loading for 1 hour could activate ERK5 phosphorylation. Both osteoblast and FSS could activate the phosphorylation of ERK1/2 (P0.001). The osteogenic differentiation index of FSS group: ALP activity. The content of calcium nodule and the expression of Runx-2Col 1 were higher than those of control group (P 0.01). The expression of XMD8-92FSS XMD8-92 in osteoblastic induction fluid was significantly higher than that in control group (P < 0.05). The above indexes of osteogenic differentiation in XMD8-92 group were also higher than those in XMD8-92 group without XMD8-92. The indexes of osteogenic differentiation in the FSS osteogenic induction solution group were higher than those in the pure osteogenic induction fluid group or the FSS group (P 0.05). Conclusion: FSS can promote the osteogenic differentiation of ERK1/2 by stimulating phosphorylation of BMSCs. XMD8-92, a specific inhibitor of ERK5, could promote the osteogenic differentiation of BMSCs. Phosphorylation of ERK5 inhibits the osteogenic differentiation of BMSCs.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R580

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