【作者】 丁慧华；

【导师】 史轶蘩；

【作者基本信息】 中国协和医科大学 ， 临床医学， 2008， 博士

【摘要】 目的:肥胖已经成为危害人类健康的全球性健康问题,与2型糖尿病、高血压、心脑血管疾病、部分肿瘤等多种疾病的发生、发展密切相关。据世界卫生组织最新报道,全球肥胖及超重人口已达20亿,到2015年预计该数字将增加到30亿。肥胖是能量摄入与消耗平衡破坏的结果,而其核心的病理生理学机制则是脂肪细胞数量的不断增加与体积的不断增大使得体内脂肪组织含量不断增加,最终导致肥胖。上述过程通过前脂肪细胞的增殖与分化实现,因此对前脂肪细胞增殖与分化的研究在肥胖发生机制的研究中具有举足轻重的地位。锌α₂糖蛋白（ZAG）是近年来新发现的脂肪细胞因子之一,最初在肿瘤恶液质患者中发现该蛋白在恶液质的发生中发挥一定作用。进一步的动物研究与体外细胞研究证实,ZAG具有强烈的促脂肪分解作用,提示该蛋白有望在肥胖治疗中发挥一定的作用。然而,目前为止,尚无有关ZAG对前脂肪细胞增殖与分化影响的研究报道,我们试图就ZAG对3T3-L1小鼠前脂肪细胞增殖与分化的影响展开研究。为深入阐明ZAG对3T3-L1细胞增殖与分化影响的可能机制,我们进一步选择脂肪细胞内脂质代谢关键酶:脂肪酸合成酶（fatty acidsynthase,FAS）与激素敏感脂肪酶（hormone sensitive lipase,HSL）作为研究目标,探讨ZAG对FAS和HSL基因表达的影响。方法:1.利用阳离子脂质体法向3T3-L1小鼠前脂肪细胞转染mZAG表达质粒（pcDNA3.1（-）-mZAG）,建立ZAG过表达的体外细胞培养模型。2.以MTT法观察不同浓度mZAG表达转染后不同时间,ZAG对3T3-L1细胞的增殖的影响。3.采用含胰岛素、地塞米松与IBMX的分化培养基诱导分化3T3-L1细胞,于分化不同阶段向细胞内转染不同浓度mZAG表达质粒。转染后第二天,以油红O染色与染料提取,以及细胞内甘油三酯测定法测定细胞内脂质含量,从而观察ZAG对3T3-L1前脂肪细胞分化的影响。4.分化不同阶段向3T3-L1细胞内转染不同浓度mZAG表达质粒,转染后第二天收集细胞标本,提取总RNA并逆转录为cDNA,采用实时荧光定量PCR对细胞内FAS与HSLmRNA表达水平进行检测,以研究ZAG对FAS及HSLmRNA表达水平的影响。5.分化不同阶段向3T3-L1细胞内同时转染不同浓度mZAG表达质粒与相同浓度分别含FAS与HSL基因启动子序列的萤光素酶报告基因质粒,转染后第二天采用双萤光素酶报告基因监测系统测定FAS与HSL基因启动子活性,以研究ZAG对FAS及HSL基因启动子活性的影响。结果:1.ZAG对3T3-L1细胞的增殖具有刺激作用,该刺激作用具有剂量与时间依赖性,最大刺激作用发生与最高浓度转染组转染后144小时,为对照组的143.4±12.1%（p＜0.01）。2.ZAG抑制3T3-L1细胞的分化,油红O染色及细胞内甘油三酯测定结果显示,mZAG过表达组细胞内脂质含量相对于对照组显著下降。3.ZAG抑制FAS基因的表达。不同浓度mRNA质粒于分化不同时间转染细胞后,细胞内FAS mRNA表达水平相对于对照组均显著下降,FAS启动子活性也受到显著抑制。4.ZAG抑制HSL基因的表达。不同浓度mRNA质粒于分化不同时间转染细胞后,细胞内HSL mRNA表达水平相对于对照组均显著下降,HSL启动子活性也受到显著抑制。结论:本研究成功建立了ZAG过表达体外细胞培养模型,并且以此为基础,成为第一例探讨ZAG对3T3-L1小鼠前脂肪细胞增殖与分化的影响的研究。结果显示,ZAG对3T3-L1小鼠前脂肪细胞的增殖具有促进作用,该促进作用具有浓度与时间依赖性;ZAG对3T3-L1小鼠前脂肪细胞的分化具有抑制作用,这一抑制作用可能是通过ZAG抑制细胞内脂肪代谢关键酶FAS与HSL基因的表达实现的。更多还原

【Abstract】 Objective:Obesity is an epidemic disease that threatens to inundate health care resources by increasing the incidence of diabetes,heart disease,hypertension,and cancer.It has reached epidemic proportions globally,with more than 2 billion adults overweight.The etiology or cause of obesity is an imbalance between the energy ingested in food and the energy expended.The excess energy is stored in fat cells that enlarge and/or increase in number.It is this hyperplasia and hypertrophy of adipocytes that is the pathological lesion of obesity.The research on the process of preadipocytes proliferation and differentiation will shed light onto the pathogenesis of obesity.Zincα-2 glycoprotein (ZAG),a 43-kDa protein,is a newly established adipokine,which was initially found to play a role in cachexia.In vivo and in vitro studies further implicated that ZAG significantly stimulates lipolysis,indicating that ZAG is a promising compound which can be used in obesity management.However,no one has ever reported the effects of ZAG on the proliferation and differentiation of preadipocytes.Since proliferation and differentiation of preadipocytes is the key pathophysiological factor of obesity,we sought to investigate the effects of ZAG on the proliferation and differentiation of a widely used adipocyte cell line,the 3T3-L1 cell line.Further more,we completed assays on ZAG overexpression of fatty acid synthase and hormone sensitive lipase gene expression in order to further explore the mechanism underlying the effects of ZAG on proliferation and differentiation of 3T3-L1 preadipocytes.Methods:1.In order to establish a ZAG overexpression cell culture model,mouse ZAG expression plasmids(pcDNA3.1(-)-mZAG) containing mouse ZAG cDNA were cloned and transfected into 3T3-L1 preadipocytes by lipofectamine.2.In order to study the effects of ZAG on the proliferation of 3T3-L1 preadipocytes, after certain hours of various amount of mZAG expression plamids transfection, 3T3-L1 cell proliferation was determined by MTT spectrophotometry.3.In order toinvestigate the effects of ZAG on the differentiation of 3T3-L1 preadipocytes,differentiation of 3T3-L1 preadipocyts were induced by insulin, dexamethasone and IBMX.Various amounts of mZAG expression plasmids were transfected into the differentiated 3T3-L1 cells during various periods throughout differentiation.Two days after transfection,red oil O staining and extracts as well as intracellular triglyceride assays were used to measure intracellular lipid content, which served as a morphological marker of adipocyte differentiation.4.In order to analyze the mechanism underlying the the effects of ZAG on proliferation and differentiation of 3T3-L1 preadipocytes,mRNA expression of FAS and HSL were measured by measn of reverse transcription quantitative polymerase chain reaction(RT-qPCR).Various amounts of mZAG expression plasmids were transfected into differentiated 3T3-L1 cells during various periods through out differentiation.Two days after transfection,total RNA levels were extracted from 3T3-L1 cells.5.In order to analyze the mechanism underlying the effects of ZAG on proliferation and differentiation of 3T3-L1 preadipocytes,promoter activity of FAS and HSL gene were measured by means of dual luciferase reporter assay system.Various amounts of mZAG expression plasmids and certain amounts of luciferase expression plasmid containing the human adipose tissue FAS and HSL promoter were transfected into the differentiated 3T3-L1 cells during various periods through out differentiation. Measurements were performed two days after transfection.Results:1.ZAG stimulated 3T3-L1 cell proliferation in a dose and time dependent manner.The maximum extend of stimulation(143.4±12.1%) occurred 144 hours after transfection at the highest dose plasmid group.2.ZAG inhibited the differentiation of 3T3-L1 cell.Intracellular lipids content measured by red oil O extracts and triglyceride assays indicate that intracellular lipids contents of mZAG overexpression groups were significantly decreased compared with the control group at various periods throughout differentiation.3.ZAG inhibited the expression of FAS.FAS mRNA level measured by RT-qPCR and FAS promoter activity measured by dual luciferase reporter assay system both imply that the expression of FAS in mZAG overexpression groups were significantly attenuated compared with control group.4.ZAG inhibited the expression of HSL.HSL mRNA level measured by RT-qPCR and HSL promoter activity measured by dual luciferase reporter assay system both imply that expression of HSL in mZAG overexpression groups were significantly attenuated compared with control group. Conclusion:We have successfully established a ZAG overexpression cell culture model Based on this model,we were able to be the first group to investigate the effects of ZAG on the proliferation and differentiation of preadipocytes.ZAG stimulated 3T3-L1 cell proliferation in a dosage and time dependant manner,and at the same time,it inhibited the differentiation of the 3T3-L1 cell line.The mechanism underlying the inhibition effect of ZAG on cell differentiation might be due to the attenuation of the expression of two key enzymes in the metabolism of lipids(FAS and HSL) by ZAG.更多还原