【作者】 田振华；

【导师】 马向华； 沈捷；

【作者基本信息】 南京医科大学 ， 内分泌科， 2009， 硕士

【摘要】 世界肥胖人口的数量正在迅速膨胀,2005年世界卫生组织(WHO,world health organization)公布,全球有2千万5岁以下儿童和16亿大于15岁人群,体重超重,4亿成人患有肥胖。WHO预言,到2015年,成人中将有23亿超重和7亿肥胖患者。2005年我国成人超重率为22.8%,肥胖率为7.1%,估计人数分别为2.0亿和6000多万。肥胖是动脉粥样硬化、高血压、2型糖尿病、癌症的高危因素,严重影响人类的生活质量,成为政府财政负担的重大公共卫生问题。肥胖与非特异性炎症的关系是近年来解读肥胖发生机制的重要进展。1993年Hotamisligil等人首次发现脂肪细胞可表达肿瘤坏死因子TNF-α,且肥胖动物的脂肪细胞TNF-α表达明显增强,从而首次将肥胖与炎症联系在一起。Weisberg和Xu首先报道了在肥胖、胰岛素抵抗脂肪组织,单核巨噬细胞浸润数量增加;手术或者饮食运动减肥后,以及胰岛素增敏剂罗格列酮干预后,白色脂肪组织单核巨噬细胞浸润减少,脂肪组织和血浆促炎标志物表达减少;提示单核巨噬细胞在肥胖的发生发展过程中具有重要作用。肥胖状态时浸润白色脂肪组织的单核巨噬细胞主要来自血液的单核细胞,也可由脂肪前体细胞转化而来。脂肪前体细胞在适当的体内外刺激下可转化成类巨噬细胞。脂肪细胞和巨噬细胞均分泌炎症因子,而且这两种细胞之间可以通过旁分泌机制相互影响激活,并通过炎性因子进行细胞串话(cross talk)。肥胖时表达明显升高的IL-6、TNF-α等炎性因子,通过旁分泌和自分泌作用于脂肪组织内共存的脂肪细胞和巨噬细胞,加重脂肪组织炎症;也可分泌至血液,作用于脂肪外组织,参与全身的炎症反应,形成恶性循环。Toll样受体4(Toll-likereceptor4,TLR4)是发现最早Toll样受体(TLR)亚型之一,由于TLR4在识别LPS及介导炎症反应信号转导中有重要作用,它在感染性疾病中的作用也越来越多地引起人们关注。研究发现TLR4不仅是LPS的主要受体,还能识别FFA、TNF-α、IL-6等多种分子。目前已知TLR4介导的信号转导包括MyD88依赖性和非依赖性途径。MyD88依赖性途径主要介导NF-κB及JNK的活化,促发炎性细胞因子产生。而MyD88非依赖性途径主要负责LPS诱导的IFN诱导性蛋白1(0IFN-indueible protein 10)、糖皮质激素终止反应基因16(glucocortcoid attenuated response gene16, GARG-16)、INF调节基因1(INF-regulated gene 1,IRG-1)表达以及树突状细胞(DC)成熟。而显然该途径的信号调节作用的意义更大。TLR4信号通路是肥胖引起血管炎症和胰岛素抵抗发生的关键因素。肥胖时增加的营养性脂肪酸可激活脂肪细胞及巨噬细胞的TLR4信号转导,而在TLR4缺失的情况下,诱导炎症信号转导的脂肪细胞或该组织的脂肪酸的产生减少。TLR4基因敲除小鼠可避免高脂饮食诱导的胰岛素抵抗,这主要是因为降低了肝脏和脂肪中炎性基因的表达。也就是说,TLR4是营养物质、脂质和炎症之间的分子联系枢纽,以使天然免疫系统参与调节能量平衡和胰岛素抵抗,用于应答营养环境的改变。本课题三部分实验分别通过IL-6对细胞炎症动态改变中TLR4受体表达、分布和其相关信号转导通路的影响,进而研究TLR4受体及下游炎症转导通路在单核/巨噬细胞激活状态下的特性,初步探讨了炎症在肥胖发生中的可能作用及机制。在第一部分实验中,我们通过研究不同浓度IL-6对体外培养的人单核/巨噬细胞系THP-1细胞膜表面表达TLR4的影响,以及对THP-1细胞周期细胞增殖的影响,同时观察特异性蛋白磷酸化的改变、并用特异的信号通路抑制剂干预,观察上述效应的相应变化。结果显示:IL-6干预THP-1细胞后,细胞表面TLR4受体激活,表达增加,同时THP-1细胞的增殖增加,S期THP-1细胞增多,其作用机制与细胞内Myd88、JNK通路激活有关。在第二部分实验中,通过研究人单核/巨噬细胞系THP-1与人原代脂肪细胞共培养,了解IL-6对细胞炎症动态改变时TLR4受体表达的量及分布、对细胞周期改变及其相关信号转导通路的影响,结果发现共培养体系建立后THP-1细胞表面TLR4受体表达增加,同时与对照组比较THP-1的增殖增加;加入IL-6干预后TLR4受体表达进一步增加、THP-1的增殖更加显著;而加入JNK抑制剂后,这种增殖受到抑制。第三部分实验中,通过从外周血中分选人原代单核细胞,初步探讨了棕榈酸棕榈酯对其增殖的影响,结果发现棕榈酸棕榈酯可明显促进人原代单核细胞的增殖,为下一步人原代脂肪细胞和人原代单核细胞共培养提供了技术平台的同时,初步探讨了脂质代谢异常与炎症的关系。本课题首次使用人单核/巨噬细胞系THP-1、及人单核/巨噬细胞系THP-1、人原代脂肪细胞两者共培养为研究平台,通过体外实验证实TLR4信号通路及下游炎症转导通路的激活在肥胖的发生机制中发挥重要作用,探讨了炎症在肥胖症发生中的可能作用及机制,为揭示肥胖症发病机制,预防、干预肥胖及肥胖相关性疾病的发生发展提供依据。根据实验证据展示阻断或调节TLR4信号通路及下游炎症转导通路的传导过程有望成为干预治疗肥胖症发生发展的新靶点;同时本研究也为肥胖的早期诊断、预后判断以及指导治疗,提供一定的实验和理论依据。更多还原

【Abstract】 Obesity has been identified as an epidemic disease in the world for more than two decades. Nowadays, the prevalence of obesity is increasing rapidly throughout the world. The reported from WHO, it is believe that, among the adult, there will be 2.3 billion people suffer from overweight and 700 million people suffer from obesity in 2015.In our country, the situation of the obesity and overweight has been serious with the ratio of 7.1% for obesity and 22.8% for overweight in 2005.The number was about 60 million and 200 million respectively at that time. Diseases involving obesity such as diabetes, cardiovascular diseases (including hypertension, coronary heart disease, and apoplexy) have grown up to the leading killer in our country and become to a heavy health burden for our government.Obesity is a situation compounding excessive fat or is abnormal in adipose tissue distribution for the body. Systemic chronic inflammation has been proposed to have an important role in the pathogenesis of obesity. Lipocyte can secrete the protein cancer putrescence gene—tumor necrosis factor-α(TNF-α), and the bigger the BMI is, the more the TNF-αwill be secreted, which was found by many researchers including Hotamisligil in 1993, causing the correlation between obesity and inflammation.It has been shown that biomarkers of inflammation, such as TNF-α, IL-6, and C-reactive protein (CRP), are present at increased concentrations in individuals who are insulin resistant and obese, and these biomarkers predict the development of Type 2DM. Furthermore, some research showed that obesity is characterized by macrophage accumulation in white adipose tissue, which has added another dimension to our understanding for the development of adipose tissue inflammation in obesityMacrophages are a major cellular component of all inflammatory sites, and the products of activated macrophages, including the classical pro-inflammatory cytokines such as TNF-α, interleukin (IL)-1, and IL-6, are central to the initiation and maintenance of inflammation. Adipose tissue macrophages (ATMs) are likely to contribute to the production of several of the adipokines as we discussed before. A causative role of ATMs in obesity-associated insulin resistance has been recently supported by many studies showing that inhibition of macrophage recruitment in obesity ameliorates the insulin resistance seen in animal models.Refer to the molecular mechanism of obesity involve inflammation, studies mainly focused on the Toll-like receptor (TLR) family, through which the microorganisms induce the transcription of proinflammatory genes with pathogen pattern recognition receptors. The most studied TLR ligand is the lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, recognition of which requires TLR4. LPS stimulate the macrophages and then initiates a complex signaling cascade reaction. Recruitment of adaptor proteins such as MyD88 to the TLR4 receptor activates a signaling cascade, which culminates in nuclear translocation of the transcription factor complex, nuclear factor NF-κB. Many other pathways, acting in series or in parallel, are required for the complete response to LPS. TLR4 related signal pathway is the key factor for the vascular inflammatory and insulin resistance caused by obese.Our study partially explained the detailed molecular mechanism of TLR4 signaling pathway in the inflammatory induced obesity. In the part one study, we try to explore the role of TLR4 signal transduction system in IL-6 treated human Macrophages—THP-1 cell line, The observation for the expression of TLR4 on cell membrane, the cell cycle and proliferation effect for THP-1, and the change for the specific protein phosphorylation have been down in both with or without specific inhibitors groups. The results show that: when treated with IL-6, the TLR4 receptor on THP-1 cell’s surface was activated, the expression increased, and the proliferation of THP-1 cells was increased too, which may be related to the activation of Myd88/ JNK pathway .In the part two study, we studied the role of TLR4 signal transduction system in the co-cultured human monocyte/macrophage cell line THP-1 and human adipose cell system. The expression and distribution of TLR4 receptor during dynamic inflammatory change induced by IL-6, and the effect on cell cycle and associated signal transduction pathway of THP-1 cell have been observed. We found that after the establishment of co-culture system, expression of TLR4 receptor on THP-1 cell surface was increased; at the same time, the proliferation of THP-1 cell was also increased compared with the control group. By adding IL-6, there was a further increase in the expression of TLR4 receptor and the proliferation in THP-1 cell. On the other hand, these changes mentioned above were inhibited significantly after added the JNK inhibitor.In the third part, we explore the effect of palmityl palmitate on the proliferation of human peripheral blood monocytes. Results showed that palmityl palmitate can significantly promote human primary monocytes proliferation in a dose and time-dependent manner, whose maximum effect appears at 20μM and 24 hours. These findings not only provide us the co-culture technology platform of human primary fat cells and human primary monocytes, but also elaborate the abnormal lipid metabolism and inflammation preliminary.For the first time, we used culture of human monocyte/macrophage cell line THP-1, and co-culture of THP-1 and human primary adipose cells as the platform, confirmed that TLR4 signaling pathway and its downstream inflammatory pathway play an important role in the pathogenesis of obesity, and discussed the possible role of inflammation in the cause of obesity. It is useful for us to reveal the pathogenesis of obesity, and provide more effect methods for the prevention, intervention of obesity and obesity-related diseases. According to our evidence, adjust or block the TLR4 signaling pathway and its downstream inflammation pathway is expected to become the new target for the treatment of obesity. At the same time, our study may help us for the early diagnosis, prognosis and guide treatment of obesity.更多还原