本文選題：骨質疏松癥　切入點：骨髓間充質干細胞　出處：《山東農業(yè)大學》2017年碩士論文

【摘要】：骨質疏松癥(Osteoporosis)是一種常見的骨代謝性疾病,隨著社會的老齡化,該病的患病率正日益上升,嚴重影響著人類的健康。由于老年男性骨質疏松發(fā)病率的提高,雄激素對骨的作用機制成為人們關注的熱點。成骨細胞、破骨細胞、骨細胞、骨髓間充質干細胞表面均有雄激素受體,雄激素對成骨細胞的調控是直接通過成骨細胞上的雄激素受體實現(xiàn)的。骨髓間充質干細胞(Bone Marrow Mesenchymal Stem Cells,BMSCs)是可以向不同類型的細胞發(fā)生轉化的,其轉化命運由細胞外信號分子調控。其中,細胞因子Chemerin其受體之一CMKLR1能夠介導免疫、炎癥、代謝、生殖及癌癥方面的疾病。Chemerin與其受體CMKLR1協(xié)調作用可以促進骨髓間充質干細胞向脂肪細胞轉化,而CMKLR1介導的信號通路在骨形成中的作用至今沒有報道,本文主要闡釋了雙氫睪酮與CMKLR1信號通路對骨形成的影響。為了闡明DHT/CMKLR1信號通路對小鼠體骨髓間充質干細胞分化能力的影響以及對小鼠骨組織代謝的影響,本課題主要從體內試驗和體外試驗進行了探究。對于體內試驗,我們對出生25天的C57雌鼠進行了分組,分為對照組、DHT組和DHT+α-NETA組,每組5只,處理21天后取股骨和脛骨進行骨組織micro-CT掃描分析,檢測骨礦物質密度、骨小梁數(shù)和骨體積分數(shù);用熒光定量的方法檢測骨形成相關因子ALP、Runx2和Col1a2的表達,探究CMKLR1被拮抗后DHT對骨代謝的調控。對于體外試驗主要是分離培養(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及通過瞬時轉染CMKLR1 siRNA來沉默CMKLR1,降低CMKLR1與Chemerin的結合,觀察BMSCs的分化,通過堿性磷酸酶染色的方法檢測ALP的活性,通過茜素紅染色的方法檢測鈣結節(jié)的形成情況,通過熒光定量的方法檢測成骨相關基因ALP、Runx2和Osterix表達的變化,用Western blot的方法檢測Runx蛋白的變化。結果表明,在體內試驗中DHT增加了骨密度、骨小梁數(shù)和骨體積分數(shù),骨形成相關因子ALP、Runx2和Col1a2的表達也都上調;注射α-NETA后,各項指標都有所下降。體外培養(yǎng)誘導BMSCs的試驗也顯示,CMKLR1缺失后堿性磷酸酶的活性降低,鈣結節(jié)形成減少,成骨相關基因的表達和Runx2蛋白都下調。因此,DHT可以促進骨髓間充質干細胞向成骨細胞分化,并促進成骨細胞的礦化,CMKLR1基因缺失則會抑制DHT對骨髓間充質干細胞的成骨分化作用,可能是通過調節(jié)成骨分化相關的maker基因Runx2、ALP等實現(xiàn)的,CMKLR1基因缺失,Chemerin/CMKLR1信號通路受到抑制,影響了雄激素對骨代謝的作用。
[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.
【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R580

【相似文獻】

相關期刊論文 前10條

1 高敏四;;色譜-分光光度法測定雙氫睪酮[J];國外醫(yī)藥.合成藥.生化藥.制劑分冊;1987年03期

2 宓志鈞,張瑞金,張美云,丁霆;血漿雙氫睪酮(經紙層析)放射免疫分析的研究[J];中華內分泌代謝雜志;1985年01期

3 史建棟，馬成禹;5α－雙氫睪酮B環(huán)衍生物的合成[J];中國藥物化學雜志;1996年02期

4 孔天翰,范天生,楚憲襄,韓雪飛,方智慧,任清雨;針刺“足三里”對大鼠血漿睪酮、雙氫睪酮含量的影響[J];河南醫(yī)科大學學報;1991年01期

5 沈彬;杜怡峰;相媛媛;叢琳;;5α-雙氫睪酮對解聚素金屬蛋白酶9表達水平的影響[J];腦與神經疾病雜志;2013年06期

6 應俊,姚德鴻,張慶華;血清睪酮、游離睪酮、雙氫睪酮濃度與男性年齡關系的研究[J];中國男科學雜志;2000年01期

7 江洪濤;陳昭典;;前列腺組織雙氫睪酮結合容量與年齡的關系及意義[J];中國男科學雜志;2007年01期

8 鄭曉春,陳淑英,鄭松柏,楊儉英,甘潔民,金健華,江魚;中老年男性血清睪酮、游離睪酮、雙氫睪酮和性激素結合球蛋白含量的變化[J];中國男科學雜志;2003年04期

9 陳君;梁瓊麟;李琿;羅國安;王義明;;利用GC/MRM檢測去勢大鼠前列腺中睪酮、雙氫睪酮和脫氫表雄酮[J];高等學�；瘜W學報;2008年11期

10 李鐘;莫曾南;楊小麗;龐友紅;黃逢雨;宣強;;雙氫睪酮對人前列腺癌細胞株PC-3細胞生長影響的體外研究[J];臨床泌尿外科雜志;2007年09期

相關會議論文 前4條

1 叢琳;杜怡峰;;5α-雙氫睪酮對解聚素金屬蛋白酶9表達水平的影響[A];山東省2013年神經內科學學術會議暨中國神經免疫大會2013論文匯編[C];2013年

2 崔慧先;李楠;李莎;康林;杜鵑;;雙氫睪酮對SAMP8小鼠海馬CA1區(qū)突觸結構影響的研究[A];中國解剖學會2011年年會論文文摘匯編[C];2011年

3 湯元杰;孫穎浩;;5α-雙氫睪酮對前列腺癌LNCaP細胞鈣離子移動和細胞生長的影響[A];第十五屆全國泌尿外科學術會議論文集[C];2008年

4 唐悅清;孫曉文;夏術階;;雙氫睪酮-蛋白水解靶向嵌合體對前列腺癌LNCaP細胞雄激素受體的降解作用[A];第十五屆全國泌尿外科學術會議論文集[C];2008年

相關重要報紙文章 前1條

1 劉文山;藥物可輔助毛發(fā)再生[N];大眾衛(wèi)生報;2005年

相關博士學位論文 前1條

1 潘文森;雙氫睪酮對AD所致MCI期SAMP8雄性小鼠空間學習記憶及海馬突觸可塑性的影響[D];河北醫(yī)科大學;2015年

相關碩士學位論文 前3條

1 趙春節(jié);雙氫睪酮對強直性脊柱炎成纖維細胞的成骨作用研究[D];廣西醫(yī)科大學;2014年

2 李楠;雙氫睪酮對SAMP8小鼠海馬突觸結構影響的研究[D];河北醫(yī)科大學;2011年

3 許丹;AR、SRD5A2基因突變與46,XY性腺發(fā)育不良相關性研究[D];復旦大學;2013年

，

本文編號：1694192