發(fā)布時(shí)間：2018-02-26 14:17

  本文關(guān)鍵詞： 原代前脂肪細(xì)胞 Wnt/β-catenin信號通路 LiCl Chemerin TNF-α　出處：《山東師范大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目前，越來越多的人患有肥胖癥，其可引發(fā)多種并發(fā)癥，如冠心病、動(dòng)脈粥樣硬化、糖尿病、高血壓、高血脂及腫瘤等疾病，嚴(yán)重威脅著人類健康。因此，研究肥胖的發(fā)生機(jī)制和防治措施已成為21世紀(jì)生命科學(xué)和醫(yī)學(xué)研究的迫切任務(wù)。 白色脂肪組織的過度積累引發(fā)肥胖，而脂肪細(xì)胞的分化在肥胖發(fā)生過程中扮演了重要角色，涉及復(fù)雜的網(wǎng)絡(luò)分子調(diào)控。Wnt/β-catenin信號通路、chemerin/CMKLR1信號通路及TNF-α均參與脂肪細(xì)胞分化的調(diào)控過程。其中，Wnt/β-catenin信號通路通過下調(diào)C/EBPs和PPARγ的表達(dá)水平，抑制成脂分化。Chemerin作為新近發(fā)現(xiàn)的脂肪因子，通過自分泌和旁分泌的方式調(diào)節(jié)脂肪細(xì)胞分化。有研究表明，分別抑制chemerin及其受體CMKLR1的表達(dá)后，可使脂肪細(xì)胞的分化過程明顯受阻；同時(shí)，chemerin可降低GSK-3β的磷酸化水平，激活GSK-3β，而后者是影響Wnt/β-catenin信號通路中β-catenin是否降解的關(guān)鍵因子。由此推測，Wnt/β-catenin信號通路與chemerin信號分子在調(diào)控成脂分化過程中存在串話。然而，其串話機(jī)制還未明確，尤其在脂肪細(xì)胞分化過程中，激活Wnt/β-catenin信號通路對chemerin的表達(dá)有何影響？其影響機(jī)制是什么？目前未見研究報(bào)道。另外，TNF-α作為一種炎癥因子，也通過多種機(jī)制抑制成脂分化，而它與Wnt/β-catenin及chemerin/CMKLR1信號通路之間的關(guān)系還不清楚。 本實(shí)驗(yàn)以WT小鼠、TNF-α-/-小鼠、Leprdb/db小鼠及DT小鼠為實(shí)驗(yàn)材料，使用Wnt/β-catenin信號通路的激活劑LiCl，干預(yù)四種基因型小鼠前脂肪細(xì)胞的分化過程，建立Wnt/β-catenin信號通路激活模型，通過實(shí)時(shí)熒光定量PCR和免疫印跡技術(shù)，檢測成脂分化中chemerin及成脂相關(guān)因子的表達(dá)變化，并分析TNF-α缺失情況下，對LiCl引起chemerin表達(dá)變化的影響。實(shí)驗(yàn)結(jié)果如下： 1.誘導(dǎo)WT小鼠前脂肪細(xì)胞分化，確定成脂分化過程中四個(gè)關(guān)鍵時(shí)期，即未誘導(dǎo)時(shí)期（誘導(dǎo)前）、誘導(dǎo)早期（誘導(dǎo)3天后）、誘導(dǎo)中期（誘導(dǎo)7天后）及誘導(dǎo)晚期（誘導(dǎo)14天后）。 2.形態(tài)學(xué)觀察發(fā)現(xiàn)，20mM LiCl顯著抑制四種基因型小鼠前脂肪細(xì)胞的分化，且抑制效果無明顯差異。另外，TNF-α-/-小鼠脂肪細(xì)胞的成脂誘導(dǎo)率最高，約80%，WT小鼠次之，DT小鼠再次之，Leprdb/db小鼠最低，約40%。 3.實(shí)時(shí)熒光定量PCR結(jié)果表明，使用LiCl干預(yù)四種基因型小鼠前脂肪細(xì)胞分化過程中，與非干預(yù)組相比，成脂相關(guān)因子C/EBPβ、C/EBPα、PPARγ2及adiponectin的基因表達(dá)水平均下調(diào)，Wnt/β-catenin信號通路關(guān)鍵分子β-catenin的基因表達(dá)水平上調(diào)，而chemerin的基因表達(dá)水平于分化早期下調(diào)，分化中期和晚期均上調(diào)。另外，TNF-α缺失可上調(diào)成脂分化早期chemerin基因的表達(dá)，并且對LiCl通過正反饋調(diào)節(jié)機(jī)制引起的chemerin基因表達(dá)的上調(diào)起促進(jìn)作用。 4.免疫印跡結(jié)果表明，使用LiCl干預(yù)四種基因型小鼠前脂肪細(xì)胞分化過程中，與非干預(yù)組相比，adiponectin蛋白的表達(dá)水平顯著下調(diào)，Wnt/β-catenin信號通路關(guān)鍵分子β-catenin蛋白的表達(dá)水平略上調(diào)，，而chemerin蛋白的表達(dá)水平于分化早期下調(diào)，分化中期和晚期均無明顯變化。 上述結(jié)果表明，在脂肪細(xì)胞分化過程中，Wnt/β-catenin信號通路主要在轉(zhuǎn)錄水平調(diào)控chemerin的表達(dá)，其調(diào)控作用主要表現(xiàn)在兩個(gè)階段：一是激活Wnt/β-catenin信號通路后，成脂分化早期的啟動(dòng)成脂過程被阻止，從而下調(diào)分化早期chemerin的基因表達(dá)；二是LiCl通過抑制GSK-3β的活性，激活Wnt/β-catenin信號通路后，由于正反饋調(diào)節(jié)機(jī)制上調(diào)分化中期和晚期chemerin的基因表達(dá)。同時(shí)，TNF-α參與Wnt/β-catenin信號通路對chemerin的調(diào)控過程，并且可能是負(fù)調(diào)控，其具體機(jī)制還需進(jìn)一步探討。
[Abstract]:At present , more and more people suffer from obesity , which can cause multiple complications , such as coronary heart disease , atherosclerosis , diabetes , hypertension , hyperlipidemia and tumor , which seriously threaten human health . Therefore , research on the pathogenesis and prevention of obesity has become an urgent task for the life science and medical research in the 21st century . It is suggested that the Wnt / 尾 - catenin signaling pathway and chemerin / CMLR1 signaling pathway and TNF - 偽 are involved in the regulation of the differentiation of fat cells . In this experiment , the expression of chemerin and adipogenesis - related factors was detected in WT mice , TNF - 偽 - / - mice , Leprdb / db mice and DT mice , and Wnt / 尾 - catenin signaling pathway activation model was established . 1 . Induction of differentiation of preadipocytes in WT mice to determine four critical periods in the process of adipogenesis , i.e . , induction ( pre - induction ) , induction early ( after induction of 3 days ) , induction of mid - term ( after 7 days of induction ) , and induction late ( after 14 days after induction ) . 2 . Morphological observation showed that 20 mM licl significantly inhibited the differentiation of preadipocytes in four genotypes , and the inhibitory effect was not significantly different . In addition , the adipogenic induction rate of TNF - 偽 - / - mouse adipocytes was the highest , about 80 % , and WT mice were the second , the DT mice were the lowest , Leprdb / db mice were the lowest , about 40 % . 3 . The results of real - time fluorescence quantitative PCR showed that the gene expression levels of C / EBP - 尾 , C / EBP - 偽 , PPAR緯 - 2 and PPAR緯 were down - regulated in four genotypes of mouse preadipocytes , while the gene expression level of the Wnt / 尾 - catenin signaling pathway was up - regulated during the early differentiation , mid - and late stages of differentiation . Furthermore , the deletion of TNF - 偽 could up - regulate the expression of chemerin gene in the early differentiation of lipid differentiation , and promote the up - regulation of chemerin gene expression induced by the positive feedback regulation mechanism . 4 . Western blot showed that the expression level of Wnt / 尾 - catenin signaling pathway was down - regulated and the expression level of Wnt / 尾 - catenin signaling pathway was slightly up - regulated compared with non - intervention group , while the expression level of chemerin protein was down - regulated in early differentiation , middle - stage and late stage of differentiation . The results showed that the Wnt / 尾 - catenin signaling pathway was mainly expressed in the transcriptional level of chemerin during the differentiation of fat cells .

【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R589.2

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