CMKLR1基因缺失对双氢睾酮诱导雄性小鼠骨形成的影响

发布时间：2018-04-01 05:28

本文选题：骨质疏松症　切入点：骨髓间充质干细胞　出处：《山东农业大学》2017年硕士论文

【摘要】：骨质疏松症(Osteoporosis)是一种常见的骨代谢性疾病,随着社会的老龄化,该病的患病率正日益上升,严重影响着人类的健康。由于老年男性骨质疏松发病率的提高,雄激素对骨的作用机制成为人们关注的热点。成骨细胞、破骨细胞、骨细胞、骨髓间充质干细胞表面均有雄激素受体,雄激素对成骨细胞的调控是直接通过成骨细胞上的雄激素受体实现的。骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells,BMSCs)是可以向不同类型的细胞发生转化的,其转化命运由细胞外信号分子调控。其中,细胞因子Chemerin其受体之一CMKLR1能够介导免疫、炎症、代谢、生殖及癌症方面的疾病。Chemerin与其受体CMKLR1协调作用可以促进骨髓间充质干细胞向脂肪细胞转化,而CMKLR1介导的信号通路在骨形成中的作用至今没有报道,本文主要阐释了双氢睾酮与CMKLR1信号通路对骨形成的影响。为了阐明DHT/CMKLR1信号通路对小鼠体骨髓间充质干细胞分化能力的影响以及对小鼠骨组织代谢的影响,本课题主要从体内试验和体外试验进行了探究。对于体内试验,我们对出生25天的C57雌鼠进行了分组,分为对照组、DHT组和DHT+α-NETA组,每组5只,处理21天后取股骨和胫骨进行骨组织micro-CT扫描分析,检测骨矿物质密度、骨小梁数和骨体积分数;用荧光定量的方法检测骨形成相关因子ALP、Runx2和Col1a2的表达,探究CMKLR1被拮抗后DHT对骨代谢的调控。对于体外试验主要是分离培养CMKLR1基因敲除鼠和野生型雄鼠的BMSCs,在DHT的诱导下观察BMSCs的成骨分化能力。并通过在野生型小鼠的BMSCs中添加CMKLR1的拮抗剂α-NETA及通过瞬时转染CMKLR1 siRNA来沉默CMKLR1,降低CMKLR1与Chemerin的结合,观察BMSCs的分化,通过碱性磷酸酶染色的方法检测ALP的活性,通过茜素红染色的方法检测钙结节的形成情况,通过荧光定量的方法检测成骨相关基因ALP、Runx2和Osterix表达的变化,用Western blot的方法检测Runx蛋白的变化。结果表明,在体内试验中DHT增加了骨密度、骨小梁数和骨体积分数,骨形成相关因子ALP、Runx2和Col1a2的表达也都上调;注射α-NETA后,各项指标都有所下降。体外培养诱导BMSCs的试验也显示,CMKLR1缺失后碱性磷酸酶的活性降低,钙结节形成减少,成骨相关基因的表达和Runx2蛋白都下调。因此,DHT可以促进骨髓间充质干细胞向成骨细胞分化,并促进成骨细胞的矿化,CMKLR1基因缺失则会抑制DHT对骨髓间充质干细胞的成骨分化作用,可能是通过调节成骨分化相关的maker基因Runx2、ALP等实现的,CMKLR1基因缺失,Chemerin/CMKLR1信号通路受到抑制,影响了雄激素对骨代谢的作用。
[Abstract]:Osteoporosis is a common bone metabolic disease. With the aging of society, the prevalence of osteoporosis is increasing day by day, which seriously affects human health. The mechanism of androgen action on bone has become a hot topic. There are androgen receptors on the surface of osteoblasts, osteoclasts, bone cells and bone marrow mesenchymal stem cells. Androgen regulates osteoblasts directly through androgen receptors on osteoblasts. Bone marrow mesenchymal stem cells (BMSCs) can be transformed to different types of cells. Its transformation fate is regulated by extracellular signaling molecules. CMKLR1, one of the receptors of cytokine Chemerin, mediates immunity, inflammation and metabolism. Chemerin and its receptor CMKLR1 can promote the transformation of bone marrow mesenchymal stem cells into adipocytes. However, the role of CMKLR1 mediated signaling pathway in bone formation has not been reported. The effects of dihydrotestosterone and CMKLR1 signaling pathway on bone formation were discussed. In order to elucidate the effect of DHT/CMKLR1 signaling pathway on the differentiation ability of mouse bone marrow mesenchymal stem cells and on the metabolism of bone tissue in mice. For in vivo experiment, we divided C57 female mice into two groups: control group and DHT 伪 -NETA group, with 5 rats in each group, and the control group was divided into two groups: the control group and the DHT 伪 -NETA group, and the control group was divided into two groups: the control group and the DHT 伪 -NETA group. Bone mineral density, bone trabecular number and bone volume fraction were detected by micro-CT scanning, and the expression of bone formation related factor ALP, Runx2 and Col1a2 were detected by fluorescence quantitative method. To explore the regulation of bone metabolism induced by CMKLR1 antagonized by DHT. In vitro experiments were conducted to isolate and culture CMKLR1 gene knockout mice and wild male BMSCs, and to observe the osteogenic differentiation ability of BMSCs induced by DHT. 伪 -NETA, an antagonist of CMKLR1, was added to BMSCs to silence CMKLR1 by transient transfection of CMKLR1 siRNA, which decreased the combination of CMKLR1 and Chemerin. The differentiation of BMSCs was observed, the activity of ALP was detected by alkaline phosphatase staining, the formation of calcium nodules was detected by alizarin red staining, and the expression of osteoblast associated gene ALP, Runx2 and Osterix was detected by fluorescence quantitative method. The changes of Runx protein were detected by Western blot. The results showed that DHT increased bone density, bone trabecula number and bone volume fraction, and the expression of bone formation related factors ALP, Runx2 and Col1a2 in vivo. The results of in vitro culture induced BMSCs also showed that the activity of alkaline phosphatase decreased and the formation of calcium nodules decreased after the deletion of CMKLR1. The expression of osteoblast-associated gene and Runx2 protein were down-regulated. Therefore, DHT could promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts, and promote the mineralization of osteoblasts. The loss of CMKLR1 gene could inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells by DHT. It may be that the signaling pathway of Chemerin / CMKLR1 is inhibited by regulating the maker gene Runx2ALP associated with osteogenic differentiation, which affects the effect of androgen on bone metabolism.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580

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