LiCl对不同基因型小鼠前脂肪细胞成脂分化中Chemerin表达的影响

发布时间：2018-02-26 14:17

本文关键词： 原代前脂肪细胞 Wnt/β-catenin信号通路 LiCl Chemerin TNF-α　出处：《山东师范大学》2015年硕士论文　论文类型：学位论文

【摘要】：目前，越来越多的人患有肥胖症，其可引发多种并发症，如冠心病、动脉粥样硬化、糖尿病、高血压、高血脂及肿瘤等疾病，严重威胁着人类健康。因此，研究肥胖的发生机制和防治措施已成为21世纪生命科学和医学研究的迫切任务。白色脂肪组织的过度积累引发肥胖，而脂肪细胞的分化在肥胖发生过程中扮演了重要角色，涉及复杂的网络分子调控。Wnt/β-catenin信号通路、chemerin/CMKLR1信号通路及TNF-α均参与脂肪细胞分化的调控过程。其中，Wnt/β-catenin信号通路通过下调C/EBPs和PPARγ的表达水平，抑制成脂分化。Chemerin作为新近发现的脂肪因子，通过自分泌和旁分泌的方式调节脂肪细胞分化。有研究表明，分别抑制chemerin及其受体CMKLR1的表达后，可使脂肪细胞的分化过程明显受阻；同时，chemerin可降低GSK-3β的磷酸化水平，激活GSK-3β，而后者是影响Wnt/β-catenin信号通路中β-catenin是否降解的关键因子。由此推测，Wnt/β-catenin信号通路与chemerin信号分子在调控成脂分化过程中存在串话。然而，其串话机制还未明确，尤其在脂肪细胞分化过程中，激活Wnt/β-catenin信号通路对chemerin的表达有何影响？其影响机制是什么？目前未见研究报道。另外，TNF-α作为一种炎症因子，也通过多种机制抑制成脂分化，而它与Wnt/β-catenin及chemerin/CMKLR1信号通路之间的关系还不清楚。本实验以WT小鼠、TNF-α-/-小鼠、Leprdb/db小鼠及DT小鼠为实验材料，使用Wnt/β-catenin信号通路的激活剂LiCl，干预四种基因型小鼠前脂肪细胞的分化过程，建立Wnt/β-catenin信号通路激活模型，通过实时荧光定量PCR和免疫印迹技术，检测成脂分化中chemerin及成脂相关因子的表达变化，并分析TNF-α缺失情况下，对LiCl引起chemerin表达变化的影响。实验结果如下： 1.诱导WT小鼠前脂肪细胞分化，确定成脂分化过程中四个关键时期，即未诱导时期（诱导前）、诱导早期（诱导3天后）、诱导中期（诱导7天后）及诱导晚期（诱导14天后）。 2.形态学观察发现，20mM LiCl显著抑制四种基因型小鼠前脂肪细胞的分化，且抑制效果无明显差异。另外，TNF-α-/-小鼠脂肪细胞的成脂诱导率最高，约80%，WT小鼠次之，DT小鼠再次之，Leprdb/db小鼠最低，约40%。 3.实时荧光定量PCR结果表明，使用LiCl干预四种基因型小鼠前脂肪细胞分化过程中，与非干预组相比，成脂相关因子C/EBPβ、C/EBPα、PPARγ2及adiponectin的基因表达水平均下调，Wnt/β-catenin信号通路关键分子β-catenin的基因表达水平上调，而chemerin的基因表达水平于分化早期下调，分化中期和晚期均上调。另外，TNF-α缺失可上调成脂分化早期chemerin基因的表达，并且对LiCl通过正反馈调节机制引起的chemerin基因表达的上调起促进作用。 4.免疫印迹结果表明，使用LiCl干预四种基因型小鼠前脂肪细胞分化过程中，与非干预组相比，adiponectin蛋白的表达水平显著下调，Wnt/β-catenin信号通路关键分子β-catenin蛋白的表达水平略上调，，而chemerin蛋白的表达水平于分化早期下调，分化中期和晚期均无明显变化。上述结果表明，在脂肪细胞分化过程中，Wnt/β-catenin信号通路主要在转录水平调控chemerin的表达，其调控作用主要表现在两个阶段：一是激活Wnt/β-catenin信号通路后，成脂分化早期的启动成脂过程被阻止，从而下调分化早期chemerin的基因表达；二是LiCl通过抑制GSK-3β的活性，激活Wnt/β-catenin信号通路后，由于正反馈调节机制上调分化中期和晚期chemerin的基因表达。同时，TNF-α参与Wnt/β-catenin信号通路对chemerin的调控过程，并且可能是负调控，其具体机制还需进一步探讨。
[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 - 伪 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 - 伪 - / - 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 - 伪 - / - 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 - 伪 , PPAR纬 - 2 and PPAR纬 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 - 伪 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 .

【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R589.2

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