【作者】 钟巧青；

【导师】 赵水平；

【作者基本信息】 中南大学 ， 内科学， 2007， 博士

【摘要】 背景动脉粥样硬化是严重危害人类健康的主要疾病，被称为“沉默的杀手”。动脉粥样硬化是一种炎症性疾病这一观点已得到公认。肥胖是冠心病独立的危险因素，脂肪细胞在与肥胖相关的心脏疾病以及动脉粥样硬化的形成过程中起着重要的作用。此外，脂肪组织不仅参与机体的能量稳态，而且是重要的内分泌器官，能够分泌与动脉粥样硬化相关的多种炎症因子如TNF-α、IL-6、脂联素、瘦素等。已知脂肪细胞在炎症刺激物脂多糖（Lipopolysaccharide，LPS）的作用下TNF-α、IL-6的分泌增多，因此，这种炎症状态下的脂肪细胞又称为炎性脂肪细胞。白介素-8（Interleukin-8，IL-8）是趋化因子超家族中的成员，已知内皮细胞、外周血单核细胞、平滑肌细胞以及脂肪细胞等均可以分泌IL-8，临床研究认为循环中白介素-8的浓度可能与动脉粥样硬化密切相关，但是在炎症状态下，脂肪细胞白介素-8的分泌有何改变目前知之甚少。脂肪细胞在机体胆固醇代谢中也发挥重要作用。本实验室前期研究已经证实脂肪细胞具有摄取氧化低密度脂蛋白（ox-LDL）的能力，在此过程中，过氧化物酶增殖体激活型受体γ（Peroxisome proliferator activated receptorγ，PPARγ）和CD36通路发挥着重要的作用，脂肪组织可能是体内清除ox-LDL的重要器官。胆固醇流出对脂肪细胞胆固醇代谢平衡的维持具有重大意义。B族Ⅰ型清道夫受体（SR-BI）能够介导细胞内游离胆固醇流出至高密度脂蛋白（High-density lipoprotein，HDL）。HDL具有抗炎、胆固醇逆转运等一系列抗动脉粥样硬化的作用，SR-BI在脂肪细胞高度表达，但炎症状态下脂肪细胞对ox-LDL的摄取及降解以及胆固醇流出的变化目前还不清楚，而HDL对上述环节的影响也有待于深入的研究。高密度脂蛋白（HDL）是一种脂质和蛋白含量大致均等的异质性脂蛋白，HDL的抗动脉粥样硬化作用是近年来的研究热点之一，其主要的作用机制包括逆向转运胆固醇、抗炎作用、抗氧化作用、抗血栓形成的效应以及内皮保护作用。HDL对炎性脂肪细胞分泌IL-8、炎性脂肪细胞胆固醇流出以及炎性脂肪细胞ox-LDL的摄取及降解等环节是否有直接的作用目前尚无实验证实。由于动脉粥样硬化是多因素引起的病理生理过程，因此进一步认知相关的有害因子和寻求最佳的抗动脉粥样硬化的药物将极大的改善心血管疾病诊断与治疗的现状。目的用脂多糖（Lipopolysaccharide，LPS）刺激脂肪细胞，在体外模拟脂肪细胞的炎症状态，观察炎症状态下脂肪细胞分泌IL-8的情况；探讨HDL对脂肪细胞分泌IL-8的影响及其可能的机制；观察HDL对炎症状态下脂肪细胞SR-BI表达及胆固醇流出的影响；观察HDL对炎症脂肪细胞摄取及降解ox-LDL的影响及可能的机制，进一步阐明炎症、脂肪细胞、动脉粥样硬化疾病的关系，加深对HDL抗动脉粥样硬化的了解，为临床治疗与肥胖相关的代谢性疾病提供新的治疗思路。方法培养3T3-L1脂肪细胞，至促分化成熟，用含0.2％BSA的无血清培养液饥饿疗法24小时，分别用不同浓度HDL（0μg／ml、10μg／ml、50μg／ml、100μg／ml）干预细胞，16小时后，再加入100ng／ml的LPS共同孵育6小时。收取培养上清液及细胞—70℃冻存备用。用酶联免疫吸附法（ELISA）检测各组脂肪细胞培养液中的IL-8水平；半定量逆转录多聚酶链式反应（RT-PCR）测定脂肪细胞CD36mRNA、PPARγmRNA、SR-BI mRNA的表达；液体闪烁计数器检测各组脂肪细胞氚标记胆固醇（³H-Cholesterol）的流出率。放射配基法测定各组脂肪细胞对ox-LDL的摄取和降解。结果1．LPS刺激脂肪细胞分泌IL-8，并且下调PPARγmRNA的表达；HDL呈剂量依赖性的降低炎性脂肪细胞分泌IL-8的浓度，并增加PPARγmRNA的表达。2．LPS刺激脂肪细胞使得胆固醇的流出减少，而HDL呈剂量依赖性促进炎性脂肪细胞胆固醇的流出；炎症状态下，脂肪细胞SR-BI mRNA表达下降。HDL呈浓度依赖性增加炎性脂肪细胞SR-BI mRNA表达。3．脂肪细胞可以摄取和降解¹²⁵I-ox-LDL，LPS刺激脂肪细胞使得¹²⁵I-ox-LDL的摄取和降解明显减少，其机制与PPARγmRNA和CD36mRNA的表达相关；HDL干预后炎性脂肪细胞PPARγmRNA和CD36mRNA的表达增加，对¹²⁵-ox-LDL的摄取量与降解增加。结论1．LPS可以诱发脂肪细胞的炎症反应，刺激脂肪细胞分泌IL-8，HDL通过上调PPARγmRNA的表达来抑制炎性脂肪细胞分泌IL-8，这可能是HDL具有抗炎作用的机制之一。2．炎症状态下脂肪细胞的胆固醇流出减少，而HDL通过SR-BI途径能改善炎性脂肪细胞的胆固醇流出，抑制脂肪细胞内胆固醇的蓄积。3．炎症可以损伤脂肪细胞摄取和降解ox-LDL的能力，而HDL能够增强炎性脂肪细胞对ox-LDL的摄取，可能与上调了PPARγ-CD36的表达有关。更多还原

【Abstract】 BackgroundAtherosclerosis （AS）, the so called "silent killer", is the leading disease challenging human health and responsible for the morbidity and mortality of population in developed and even partly developing countries. It is well acknowledged that AS presents an inflammatory disease. Obesity, which is associated with increased systemic inflammation, is demonstrated as an independent risk factor for coronary heart disease （CHD）. Adipose. tissue has direct connection between obesity and systemic inflammation. Adiposities’ function is of vital importance in the development of obesity-associated diseases and the process of vascular injury. Adipoeytes are, at the present, regarded as the crossroads of energy homeostasis, inflammation, and atherosclerosis. Therefore, the studies regarding adiposities’ function is accordingly highly interested by people.Adipose tissue, the largest energy reservoir in the body, can preserve redundant energy in the form as triglyceride. However, it has been shown that adipose tissue is not only an energy-regulating organ, but also an important endocrine organ in that it can secret various adipokines and chemokines, many of which might play significant roles in atherogenesis. There is an increase of metabolic diseases during the dysfunction of adipocytes, including obesity, diabetes, hypertension and dyslipidemia and so on, the majority of which can then lead to an increase of AS risk. Interleukin-8 （IL-8） is the most important cytokines produced by different cellular types in response to various stimuli. It is known as one of the super family of chemotatic factors, secreted by a serial of proatherogenic cells such as endothelial cells, peripheral blood mononuclear cells, smooth muscle cells and adipocytes. It is clinically observed that the levels of IL-8 in circulation may be relative with AS. However, it still remains to be researched about the secretion of IL-8 by adipocytes in the state of inflammation.Adipocytes also display an important role in cholesterol metabolism in the body. Because adipose tissue is found as the largest pool of free cholesterol in vivo and has a buffer action for circulatory cholesterol metabolism. Moreover, cholesterol effiux has been proven crucial for the balance of cholesterol metabolism in adipocytes.Scavenger receptor class B type I. （SR-BI）, a high density lipoprotein （HDL） receptor, can induce the effiux of free cholesterol from peripherical cells to HDL. Interestingly, SR-BI has been observed highly expressed in adipocytes. But it is still unclear about the function of SR-BI on cholesterol effiux in inflammatory adipocytes.It has been well known about the important role of oxidized low density lipoprotein （ox-LDL） in the development and progress of AS. It is regarded as one of important regulatory pathway for ox-LDL entering monocytes that peroxisome proliferators- activated receptorγ（PPARγ） induces the expression of CD36 at transcriptional level. Our pretrial work has substantiated that adipocytes have the potency to intake ox-LDL, considerably involving PPARγ-CD36 pathway, which shows adipose tissue is possibly an important organ removing ox-LDL in vivo. Therefore, it is very necessary for us to further implore the uptake and degradation of ox-LDL by adipocytes in inflammation since inflammation is regarded as a key factor in the development of AS.High density lipoprotein （HDL） is a heterogeneous protein with the almost moiety of lipid and protein and it is a hot issue on the atheroprotective role of HDL. The .related mechanisms underlying anti-AS by HDL includes cholesterol reverse transport, anti-inflammation, anti-oxidation, anti-thrombosis as well as endothelial protection. It is still unproven that there is a directed effect of HDL on the processes, including the secretion of IL-8, cholesterol effiux and the uptake and degradation of ox-LDL related inflammatory adipocytes, or not. There, it is believed to markedly improve the diagnosis and therapy of cardiovascular disease to recognize related risk factors and more efficient drug of AS, since AS is taken as a disease involving multifactor.ObjectiveWe will illustrate the relationship inflammation, adipocytes and AS for further comprehension of anti-AS by HDL, which maybe help to determine a novel therapeutic target in treatment of obesity-related metabolic diseases, via observing the secretion of IL-8 by adipocytes in inflammation, exploring the effect of HDL on the secretion of IL-8 by adipocytes and its possible mechanism, detecting the effect of HDL on the expression of SR-BI and the cholesterol effiux in adipocytes in inflammation, as well as investigating the effect of HDL on and the uptake and degradation of ox-LDL by inflammatory adipocytes and its involved mechanism.Methods3T3-L1 adipocytes were cultured and induced to differentiation and maturation. After cells were starved for 24 hours with serum-free medium containing 0.2% BSA, HDL in various concentrations （0μg/ml, 10μg/ml, 50μg/ml and 100μg/ml） were added and 6 hours later, 100 ng/ml LPS were added for co-incubation for 6 hours. Then, supernatant and cells were gathered respectively, stored in -70^C for future application. After then, IL-8 in supernatant was detected with ELISA, the mRNA expression of CD36, PPARy and SR-BI in cells was determined via RT-PCR, the effiux rates of 3H-cholesterol in cells were detected by means of liquid scintillation counter, and the uptake and degradation of ox-LDL by adipocytes in individual groups were measured by radioligand assay.Results1. LPS stimulated the secretion of IL-8 and reduced the mRNA expression of PPARγin adipocytes. HDL decreased the levels of IL-8 from inflammatory adipocytes dose-dependently and accordingly up-regulated the mRNA expression of PPARγin these cells.2. LPS decreased cholesterol effiux in stimulating adipocytes while HDL promoted the cholesterol effiux from inflammatory adipocytes in a dose-dependent manner. The mRNA expression of SR-BI declined in adipocytes with stimulated by LPS while HDL up-regulated the mRNA expression of SR-BI.3. ¹²⁵I-ox-LDL was uptaked by adipocytes and its uptake considerably declined with the stimulation of LPS, which was relative to the mRNA expression of PPAR^/and CD36. In contrast, HDL increased the uptake of¹²⁵I-ox-LDL by inflammatory adipocytes via the pathway of PPARγ-CD36.Conclusions1. LPS can induce the inflammatory response of adipocytes via increasing the secretion of IL-8 from adipocytes while HDL can inhibit IL-8 secretion in inflammatory adipocytes through the up-regulation of PPARγrnRNA expression, which maybe one of anti-inflammatory mechanisms by HDL.2. Inflammation can decrease the cholesterol efflux of adipocytes while HDL can promote the cholesterol efflux of inflammatory adipocytes via SR-BI pathway.3. Inflammation can impair the ability of adipocytes’ uptaking ox-LDL while HDL can enhance ox-LDL uptake by inflammatory adipocytes through PPARγ-CD36 pathway.更多还原