本文選題：骨質(zhì)疏松癥　切入點(diǎn)：骨髓間充質(zhì)干細(xì)胞　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：骨質(zhì)疏松癥(Osteoporosis)是一種常見的骨代謝性疾病,隨著社會(huì)的老齡化,該病的患病率正日益上升,嚴(yán)重影響著人類的健康。由于老年男性骨質(zhì)疏松發(fā)病率的提高,雄激素對(duì)骨的作用機(jī)制成為人們關(guān)注的熱點(diǎn)。成骨細(xì)胞、破骨細(xì)胞、骨細(xì)胞、骨髓間充質(zhì)干細(xì)胞表面均有雄激素受體,雄激素對(duì)成骨細(xì)胞的調(diào)控是直接通過成骨細(xì)胞上的雄激素受體實(shí)現(xiàn)的。骨髓間充質(zhì)干細(xì)胞(Bone Marrow Mesenchymal Stem Cells,BMSCs)是可以向不同類型的細(xì)胞發(fā)生轉(zhuǎn)化的,其轉(zhuǎn)化命運(yùn)由細(xì)胞外信號(hào)分子調(diào)控。其中,細(xì)胞因子Chemerin其受體之一CMKLR1能夠介導(dǎo)免疫、炎癥、代謝、生殖及癌癥方面的疾病。Chemerin與其受體CMKLR1協(xié)調(diào)作用可以促進(jìn)骨髓間充質(zhì)干細(xì)胞向脂肪細(xì)胞轉(zhuǎn)化,而CMKLR1介導(dǎo)的信號(hào)通路在骨形成中的作用至今沒有報(bào)道,本文主要闡釋了雙氫睪酮與CMKLR1信號(hào)通路對(duì)骨形成的影響。為了闡明DHT/CMKLR1信號(hào)通路對(duì)小鼠體骨髓間充質(zhì)干細(xì)胞分化能力的影響以及對(duì)小鼠骨組織代謝的影響,本課題主要從體內(nèi)試驗(yàn)和體外試驗(yàn)進(jìn)行了探究。對(duì)于體內(nèi)試驗(yàn),我們對(duì)出生25天的C57雌鼠進(jìn)行了分組,分為對(duì)照組、DHT組和DHT+α-NETA組,每組5只,處理21天后取股骨和脛骨進(jìn)行骨組織micro-CT掃描分析,檢測(cè)骨礦物質(zhì)密度、骨小梁數(shù)和骨體積分?jǐn)?shù);用熒光定量的方法檢測(cè)骨形成相關(guān)因子ALP、Runx2和Col1a2的表達(dá),探究CMKLR1被拮抗后DHT對(duì)骨代謝的調(diào)控。對(duì)于體外試驗(yàn)主要是分離培養(yǎng)CMKLR1基因敲除鼠和野生型雄鼠的BMSCs,在DHT的誘導(dǎo)下觀察BMSCs的成骨分化能力。并通過在野生型小鼠的BMSCs中添加CMKLR1的拮抗劑α-NETA及通過瞬時(shí)轉(zhuǎn)染CMKLR1 siRNA來沉默CMKLR1,降低CMKLR1與Chemerin的結(jié)合,觀察BMSCs的分化,通過堿性磷酸酶染色的方法檢測(cè)ALP的活性,通過茜素紅染色的方法檢測(cè)鈣結(jié)節(jié)的形成情況,通過熒光定量的方法檢測(cè)成骨相關(guān)基因ALP、Runx2和Osterix表達(dá)的變化,用Western blot的方法檢測(cè)Runx蛋白的變化。結(jié)果表明,在體內(nèi)試驗(yàn)中DHT增加了骨密度、骨小梁數(shù)和骨體積分?jǐn)?shù),骨形成相關(guān)因子ALP、Runx2和Col1a2的表達(dá)也都上調(diào);注射α-NETA后,各項(xiàng)指標(biāo)都有所下降。體外培養(yǎng)誘導(dǎo)BMSCs的試驗(yàn)也顯示,CMKLR1缺失后堿性磷酸酶的活性降低,鈣結(jié)節(jié)形成減少,成骨相關(guān)基因的表達(dá)和Runx2蛋白都下調(diào)。因此,DHT可以促進(jìn)骨髓間充質(zhì)干細(xì)胞向成骨細(xì)胞分化,并促進(jìn)成骨細(xì)胞的礦化,CMKLR1基因缺失則會(huì)抑制DHT對(duì)骨髓間充質(zhì)干細(xì)胞的成骨分化作用,可能是通過調(diào)節(jié)成骨分化相關(guān)的maker基因Runx2、ALP等實(shí)現(xiàn)的,CMKLR1基因缺失,Chemerin/CMKLR1信號(hào)通路受到抑制,影響了雄激素對(duì)骨代謝的作用。
[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 偽 -NETA group, with 5 rats in each group, and the control group was divided into two groups: the control group and the DHT 偽 -NETA group, and the control group was divided into two groups: the control group and the DHT 偽 -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. 偽 -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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R580

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