miR-27a通过抑制PPARγ调控巨噬细胞活化在肥胖诱导胰岛素抵抗中的作用及机制研究

发布时间：2018-06-07 06:04

本文选题：肥胖 + 胰岛素抵抗　；参考：《吉林大学》2017年博士论文

【摘要】：脂肪组织区域免疫异常引起的慢性炎症是肥胖诱导胰岛素抵抗的关键因素,巨噬细胞募集及极化是脂肪组织区域免疫异常的中心环节,而核转录因子PPARγ是巨噬细胞活化的关键调控分子。有研究表明与脂肪细胞分化相关的mi R-27a在肥胖人群的血液中表达水平明显增加,并且mi R-27a直接的下游作用靶点为PPARγ,因此我们推测,mi R-27a作为巨噬细胞活化的分子开关,通过调控PPARγ调节肥胖诱导的胰岛素抵抗。本研究通过肥胖诱导胰岛素抵抗模型小鼠、mi R-27a抑制干预的肥胖小鼠、mi R-27a慢病毒过表达小鼠以及肥大脂肪细胞和巨噬细胞共培养模型,探讨mi R-27a调控巨噬细胞募集及极化的分子机制,阐明mi R-27a参与脂肪组织区域性免疫的细胞及分子网络调控,揭示mi R-27a在肥胖诱导胰岛素抵抗中的关键作用,为肥胖诱导胰岛素抵抗提供新的干预靶点。本论文研究内容包括三个部分。第一部分探讨脂肪组织巨噬细胞活性变化在肥胖胰岛素抵抗中的作用。首先建立肥胖诱导胰岛素抵抗小鼠模型,结果显示喂养4w、8w和12w后,肥胖程度、空腹血糖、空腹胰岛素、甘油三酯、总胆固醇、低密度脂蛋白的水平均呈持续增长状态,高密度脂蛋白水平下降,并且葡萄糖耐受能力持续下降,胰岛素抵抗情况越来越明显;这一过程亦可促进血清中以及内脏脂肪组织中趋化因子MCP1和巨噬细胞M1型炎性因子TNFα的分泌;组织形态学亦可见内脏脂肪区域的脂肪细胞形态变化明显,细胞体积逐渐增大,并且伴随着大量巨噬细胞在脂肪组织中的募集和浸润,同时出现大量M1型巨噬细胞,巨噬细胞极化现象明显。结果说明巨噬细胞活化与肥胖诱导胰岛素抵抗相关。第二部分研究mi R-27a在肥胖诱导胰岛素抵抗中的关键作用及其与巨噬细胞活化的关系。结果显示,在小鼠持续给予高脂饮食建立肥胖诱导胰岛素抵抗的模型中,随着肥胖时间延长,血清中的mi R-27a分泌量从4周到12周持续升高,空腹血糖、空腹胰岛素、甘油三酯和总胆固醇等生化指标水平升高,胰岛素抵抗程度加重,提示mi R-27a可能参与胰岛素抵抗。为了证明此观点,进一步建立mi R-27a过表达和mi R-27a抑制小鼠模型,结果证明mi R-27a水平对于体重、饮食热量、体脂含量以及空腹血糖、空腹胰岛素、甘油三酯和总胆固醇的水平都有影响,mi R-27a过表达组的空腹血糖、空腹胰岛素等生化指标水平均升高,葡萄糖耐量和胰岛素耐量下降,导致胰岛素抵抗的发生,胰岛素的敏感性降低;mi R-27a过表达促进血清中和内脏脂肪组织中炎性因子(MCP1、TNFα)的分泌和释放,同时mi R-27a过表达组的脂肪组织出现大量M1型巨噬细胞。但mi R-27a抑制组的空腹血糖、空腹胰岛素等生化指标的水平均降低,改善糖耐量,增加胰岛素敏感性,缓解胰岛素抵抗;mi R-27a抑制可减少炎性因子的分泌和释放,也能明显缓解持续高脂饮食喂养引起的内脏脂肪组织巨噬细胞极化现象及局部炎症反应。说明mi R-27a在肥胖诱发胰岛素抵抗中起关键作用,其作用机制与巨噬细胞活化导致的慢性低度炎症有关。第三部分探讨mi R-27a对巨噬细胞活化调控作用的分子机制。在肥胖诱导胰岛素抵抗小鼠脂肪组织相关蛋白水平的分析显示,胰岛素代谢信号通路中的Akt在胰岛素刺激下未被激活,出现胰岛素抵抗。内脏脂肪区域PPARγ的蛋白水平下降,巨噬细胞活化相关蛋白TLR4以及p-NF-κB的蛋白水平均升高,IκBα水平下降,巨噬细胞活化通路被激活;另有下游炎性反应通路TNFR1和p-JNK的蛋白水平显著升高,刺激炎性因子的释放和分泌。细胞实验结果显示,mi R-27a overexpression质粒转染Raw264.7细胞后,PPARγ的蛋白水平下降,细胞上清液中MCP1和TNFα的水平明显增加,M2型抑炎细胞因子ARG1水平降低,但给予PPARγ激活剂罗格列酮后,细胞上清液中MCP1和TNFα的水平明显下降,ARG1水平明显增加;其次mi R-27a过表达后巨噬细胞迁移数量明显增多,同时给予罗格列酮后迁移细胞减少;另外,棕榈酸刺激使脂肪细胞肥大后,mi R-27a水平升高,促进巨噬细胞迁移,但刺激肥大的脂肪细胞转染mi R-27a inhibitor后,巨噬细胞迁移数量明显下降,在无脂肪细胞组给予棕榈酸刺激,巨噬细胞没有迁移。说明mi R-27a通过抑制PPARγ引起巨噬细胞的募集及极化,促进巨噬细胞活化。因此,本课题首次阐明mi R-27a在肥胖诱导胰岛素抵抗中起到关键作用,主要作用机制可能是肥胖过程中高表达的mi R-27a能够抑制其靶基因PPARγ的表达,进而激活NF-κB,促进炎性因子的合成及分泌,促进巨噬细胞的募集和极化,诱导肥胖胰岛素抵抗的发生,为肥胖诱导胰岛素抵抗提供新的干预靶点,亦为糖尿病预防和治疗提供实验依据和理论基础。
[Abstract]:Chronic inflammation caused by regional immune abnormality in adipose tissue is the key factor for obesity induced insulin resistance. Macrophage recruitment and polarization are central links in the regional immune abnormality of adipose tissue, and nuclear factor PPAR gamma is the key regulator of macrophage activation. Research shows that MI R-27a related to adipocyte differentiation is in fertilizer. The level of blood expression in the fat population increased significantly and the direct downstream target of MI R-27a was PPAR gamma. Therefore, we speculate that MI R-27a acts as a molecular switch activated by macrophages and regulates obesity induced insulin resistance by regulating PPAR gamma. This study inhibited the insulin resistance model mice by obesity and the MI R-27a inhibition intervention. Obese mice, MI R-27a lentivirus overexpressed mice and hypertrophic adipocyte and macrophage co culture model, explore the molecular mechanism of MI R-27a regulating macrophage recruitment and polarization, elucidate the cell and molecular network regulation of MI R-27a involved in regional immunization of adipose tissue, and reveal the key to MI R-27a in insulin resistance induced by obesity. The purpose of this study is to provide new targets for the intervention of obesity induced insulin resistance. This paper includes three parts. The first part discusses the role of the changes in the activity of macrophage in adipose tissue in the obesity insulin resistance. First, we establish a model of obesity induced insulin resistance in mice. The results showed that after feeding 4W, 8W and 12W, the degree of obesity and empty stomach was empty. The levels of blood glucose, fasting insulin, triglyceride, total cholesterol, low density lipoprotein were continuously growing, the level of high density lipoprotein decreased, and the glucose tolerance continued to decline, and insulin resistance became more and more obvious; this process also promoted the chemokine MCP1 in the serum and in the visceral adipose tissue. The secretion of M1 type inflammatory factor TNF alpha, and the morphological changes of adipocytes in the visceral adipose area are also obvious, the volume of the cells gradually increases, and the recruitment and infiltration of a large number of macrophages in the adipose tissue are accompanied by a large number of M1 macrophages, and the polarization of macrophage cells is obvious. Activation is associated with insulin resistance induced by obesity. Second the key role of MI R-27a in obesity induced insulin resistance and the relationship with macrophage activation are studied. The results show that the MI R-27a secretion in serum is prolonged with the prolonged obesity induced insulin resistance in mice. From 4 to 12 weeks, the level of fasting blood glucose, fasting insulin, triglyceride, total cholesterol and other biochemical indexes increased, and the degree of insulin resistance was aggravated, suggesting that MI R-27a may be involved in insulin resistance. In order to prove this point of view, further establishment of MI R-27a overexpression and MI R-27a inhibition mice model, the results prove that MI R-27a level is the same. The levels of body weight, diet calorie, body fat content and fasting blood glucose, fasting insulin, triglyceride and total cholesterol were all affected. The levels of fasting blood glucose, fasting insulin and other biochemical indexes in the MI R-27a overexpressed group were all elevated, glucose tolerance and insulin tolerance decreased, resulting in insulin resistance and insulin sensitivity. The overexpression of MI R-27a promotes the secretion and release of inflammatory factors (MCP1, TNF a) in serum and visceral adipose tissue, while a large number of M1 type macrophages appear in the adipose tissue of the MI R-27a overexpressed group. However, the fasting blood glucose, fasting insulin and other biochemical indexes of the MI R-27a inhibition group decrease, improving the glucose tolerance and increasing the insulin sensitivity. Alleviating insulin resistance; MI R-27a inhibition can reduce the secretion and release of inflammatory factors and significantly alleviate the polarization and local inflammatory response of visceral adipose tissue induced by continuous high fat diet. It shows that MI R-27a plays a key role in obesity induced insulin resistance, and its mechanism is associated with the activation of macrophages. The third part discusses the molecular mechanism of MI R-27a on the regulation of macrophage activation. In the analysis of adipose tissue related protein levels in insulin resistant mice induced by obesity, the Akt in insulin signaling pathway is not activated by insulin stimulation, and insulin resistance. The visceral fat region PPAR gamma The protein level of the macrophage activation related protein TLR4 and p-NF- kappa B increased, the level of I kappa B alpha was decreased, the activation pathway of macrophage was activated, and the protein level of TNFR1 and p-JNK in the downstream inflammatory pathway increased significantly and stimulated the release and secretion of inflammatory factors. The results of cell experiments showed mi R-27a overexpre. After the transfection of ssion plasmid to Raw264.7 cells, the protein level of PPAR gamma decreased, the level of MCP1 and TNF alpha in the cell supernatant increased obviously, and the level of M2 anti inflammatory cytokine ARG1 decreased, but the level of MCP1 and TNF a in the cell supernatant decreased obviously, and the ARG1 level increased obviously after the PPAR gamma activator rosiglitazone was given, followed by the giant MI. The number of macrophage migration increased significantly and the migratory cells were reduced after rosiglitazone. In addition, when palmitic acid stimulated the fat cells to hypertrophy, the level of MI R-27a increased and promoted the migration of macrophages. However, after the transfection of hypertrophic adipocytes to MI R-27a inhibitor, the number of macrophage cell migration decreased significantly and was given to brown in the adipocyte group. It is the first time that MI R-27a plays a key role in obesity induced insulin resistance, the main mechanism of which is that MI R-27a plays a key role in obesity induced insulin resistance. The main mechanism may be that the high expression of MI R-27a in the process of obesity can inhibit it in the process of obesity. The expression of target gene PPAR gamma, then activation of NF- kappa B, promotes the synthesis and secretion of inflammatory factors, promotes the recruitment and polarization of macrophages, induces the occurrence of obese insulin resistance, provides new intervention targets for obesity induced insulin resistance, and provides experimental basis and theoretical basis for the prevention and treatment of diabetes.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R58

【相似文献】