【作者】 王毅飞；

【导师】 蔡德鸿；

【作者基本信息】 南方医科大学 ， 内分泌与代谢病学， 2010， 博士

【摘要】 脂肪细胞是哺乳动物的一类重要功能细胞,在维持脂类代谢及能量代谢平衡方面发挥关键作用。前脂肪细胞分化为成熟脂肪细胞是一个复杂的过程,这一过程受到许多转录因子、细胞因子以及激素的调节。脂肪细胞分化与调控失常可导致人类多种疾病如肥胖症、2型糖尿病、脂肪肝、高脂血症及乳腺癌等。前脂肪细胞的增殖和分化过程可能是研究肥胖症发生机制的核心,同时对前脂肪细胞的增殖和分化过程的研究还可以有助于寻找减肥药物作用的靶点。因此,在细胞和分子水平上对脂肪细胞分化调控机制研究,对探讨上述重大生命和疾病过程、探索预防与治疗的新途径具有重要理论意义和应用价值。糖皮质激素(glucocorticoid, GC)诱导亮氮酸拉链蛋白(glucocorticoid induced leucine zipper, GILZ)被报道是一种糖皮质激素诱导蛋白,具有炎症抑制、促护T细胞凋亡等多项作用。糖皮质激素具有诱导细胞生长、分化和凋亡等多种功能,被认为是较好的抗炎及免疫抑制剂,已广泛用于多种疾病的治疗,如关节炎、肺气肿、哮喘、器官抑制排斥反应等。但重要的是长期的使用糖皮质激素,会加速骨的丢失导致骨质疏松,取而代之的是骨髓脂肪细胞数量的增加。报道初步显示GILZ可能通过抑制脂肪发生而拮抗糖皮质激素的作用,在GC作用当中起了负反馈作用。GILZ属于亮氨酸拉链转录因子家族成员,一些研究还表明GILZ可作为一种转录抑制因子,与NF-KB(nuclear transcription factor kappa B, NF-κB)、转录因子活化蛋白-1(activator protein-1, AP-1)直接作用后参与抑制基因表达,参与了Fas/FasL、NF-κB、Ap1、Raf21等多条信号通路的信号传递。在脂肪细胞形成过程中,作为细胞相关信号通路作用靶点的转录因子,过氧化物增殖活化受体(Peroxisome Proliferative Activated Receptor,PPARs)家族和CCAAT/增强子结合蛋白家族(CCAAT/enhance binding proteins, C/EBPs)起了重要的作用。PPARγ属于能与靶基因调节区特异DNA反应元件结合的核激素受体转录因子家族,由于启动子和拼接方式不同,有PPARγ1、PPARγ2及PPARγ3三种亚型。PPARγ2主要表达于脂肪组织,在脂肪细胞分化的早期就表达,是对脂肪细胞分化具有决定性作用的一个异构体。PPARγ2可以激活一系列脂肪代谢必需基因,包括脂蛋白脂酶(Lipoprotein lipase,LPL)、磷酸烯醇式丙酮酸羧激酶(Phosphoenolpyruvate Carboxykinase, PEPCK)、脂肪酸转运与结合蛋白(Fatty acid binding protein, FABP)、硬脂酰辅酶A去饱和酶-1(Stearoyl CoA desat urease-1, SCD-1)等,在这些基因的启动子区都发现有功能性的PPAR反应元件。另一类重要的脂肪细胞分化转录因子C/EBPs,它有C/EBPα, C/EBPβ和c/EBPδ等几种异构体,都能促进脂肪细胞分化。C/EBPα可能介导GILZ在前脂肪细胞分化中的作用,同PPARγ2可以相互激活转录,启动脂肪细胞的分化。GILZ作为一种重要的信号蛋白分子和核转录因子是否参与了脂肪细胞的分化,是否通过C/EBPα和PPARγ2的介导等分子机制还不十分清楚。因此,有关GILZ在脂肪细胞分化中的作用及其机制的阐明,将对预防和治疗肥胖及2型糖尿病具有重要的意义。本文共分三部分对GILZ在脂肪细胞分化中的作用及其机制作初步探讨。1)常规构建HA-GILZ/PcDNA3重组真核表达载体、进行细胞内表达定位；联合3-异丁基-1-甲基-黄嘌呤(3-isobutyl-1-methyl-xanthine, MIX)、地塞米松和胰岛素诱导3T3-L1前脂肪细胞,摸索建立脂肪细胞分化模型；脂质体转染3T3-L1细胞,G418筛选转染细胞,建立HA-GILZ稳定、高表达的3T3-L1前脂肪细胞细胞系。主要是为探讨GILZ的生物学功能提供基因和细胞研究工具。2)比较GILZ对前脂肪细胞增殖、前脂肪细胞内的脂质特异性着色、甘油三酯相对含量及脂肪细胞分化相关基因PPARγ2, C/EBPα, LPL和FAS的mRNA表达的影响。在细胞和基因mRNA表达水平探讨GILZ的生物学作用及机制。3)初步分析C/EBP和AP-1顺式作用元件在GILZ抑制PPARγ2启动子转录活性中的作用,在DNA转录顺式作用元件分子调控水平研究GILZ的作用机制。第一部分GILZ真核表达载体的构建、表达定位,稳定高表达GILZ的3T3-L1前脂肪细胞株的克隆目的：克隆、构建HA标签的糖皮质激素诱导亮氨酸拉链蛋白GILZ真核表达载体,观察其在细胞中表达与定位,建立3T3-L1前脂肪细胞分化模型及GILZ稳定高表达株,为研究GILZ在脂肪细胞分化中的作用提供基因和细胞工具。方法：1.采用逆转录PCR (RT-PCR)法从C3H10T1/2多能干细胞中扩增GILZ cDNA编码区,并将其重组于带有HA标签的真核表达载体PcDNA3中。BamHⅠ/XhoⅠ双酶切、DNA测序鉴定HA-GILZ/PcDNA3重组表达载体。2.采用lipofectamine 2000脂质体瞬时转染HA-GILZ/PcDNA3重组表达载体至C3H10T1/2多能干细胞；采用CaCl2方法瞬时转染HA-GILZ/PcDNA3重组表达载体至293T细胞。3.采用HA-GILZ/PcDNA3重组表达载体通过lipofectamine 2000脂质体转染3T3-L1细胞,采用G418筛选HA-GILZ稳定表达的3T3-L1前脂肪细胞细胞系。4.转染48h后,采用免疫荧光检测GILZ在C3H10T1/2多能干细胞和3T3-L1前脂肪细胞内的表达、分布定位；采用免疫印迹法检测GILZ在293T细胞和稳定转染HA-GILZ/PcDNA3的3T3-L1前脂肪细胞系内的稳定表达及分子量大小。5.用含0.5 mmol/L 3-异丁基-1-甲基-黄嘌呤(MIX)、10μg/ml胰岛素和0.25μmol/L地塞米松的脂肪细胞诱导剂(MID)诱导3T3-Ll前脂肪细胞分化,用油红0染色法观测脂肪细胞内的脂质特异性着色及测定甘油三酯相对含量。结果：1.HA-GILZ/PcDNA3重组表达载体经酶切鉴定和测序分析确认构建成功。进一步免疫荧光显示GILZ在C3H10T1/2细胞质和细胞核都有表达,但主要表达定位于细胞质。免疫印迹法检测在转染HA-GILZ/PcDNA3重组载体的293T和3T3-Ll细胞中有一特异的分子量约20kD的蛋白条带,获得了高效的表达。2.3T3-L1前脂肪细胞经MID诱导的第3天开始,细胞内可以观察到反光的脂质小滴,并随着分化的进展越来越多的细胞出现脂质小滴。到了第9天,脂肪细胞胞质内有大量圆形脂滴,而正常生长细胞无脂滴。油红0染色显示分化的3T3-L1脂肪细胞胞浆中脂滴呈桔红色。与未含MID培养基对照组相比,甘油三酯相对含量显著增加(0.139±0.022 vs 0.356±0.014,t=14.521,p=0.000)。3.将该HA-GILZ/PcDNA3重组表达载体通过lipofectamine 2000脂质体转染3T3-L1细胞后,经G418筛选的3T3-L1细胞,免疫荧光和免疫印迹均显示有GILZ高表达,并且跟C3H10T1/2细胞一样,GILZ也主要表达于细胞质。小结：1.成功构建和表达HA-GILZ/PcDNA3重组表达载体,为GILZ的功能研究提供了稳定的基因工具。2.实验表明GILZ主要定位表达于C3H10T1/2和3T3-Ll细胞质中。3.成功地诱导3T3-L1前脂肪细胞的分化,并成功建立稳定、高表达的3T3-L1细胞克隆,为GILZ的功能研究提供了稳定的细胞工具。第二部分：GILZ对前脂肪细胞增殖、脂肪细胞分化及相关基因表达的影响目的：观测GILZ对3T3-Ll前脂肪细胞增殖、分化的作用,研究GILZ对脂肪细胞分化相关标志基因PPARγ2, C/EBPα, LPL和FAS mRNA表达的影响,探讨GILZ调节3T3-L1前脂肪细胞分化的分子机制。方法：1.采用MTT法,从第1天到第11天,每隔2天检测GILZ稳定表达3T3-L1细胞细胞的增殖情况。2.油红0染色观察GILZ过表达对脂肪细胞分化和甘油三酯相对含量的影响。共分四组：1,转染对照PcDNA3,没有MID诱导；2,转染HA-GILZ/PcDNA3,没有MID诱导；3,转染对照PcDNA3, MID诱导9天；4,转染HA-GILZ/PcDNA3,MID诱导9天。3.常规构建GILZ-shRNA/pLentiLox 3.7(简称GILZ-shRNA)慢病毒重组载体、测序、lipofectamine 2000脂质体转染、293T细胞包装、感染GILZ稳定表达的3T3-L1细胞、实时荧光定量PCR (Real-time PCR)检测脂肪细胞GILZ mRNA表达。4.实时荧光定量PCR (Real-time PCR)法检测脂肪细胞分化相关标志基因PPARγ2、C/EBPα、LPL和FAS的mRNA表达。对于GILZ过表达共分二组：转染PcDNA3对照空载体；转染HA-GILZ/PcDNA3重组载体；对于GILZ沉默共分二组：感染pLentiLox 3.7空载体对照；感染GILZ-shRNA/pLentiLox 3.7重组载体。结果：1.MTT法检测结果,在细胞增殖的第1、3、5、7、9、11天,转染PcDNA3空载体对照组的OD值分别是：0.090±0.008,0.154±0.004,0.273±0.017,0.471±0.008,0.609±0.013,0.610±0.018；而稳定转染HA-GILZ的OD值分别是：0.083±0.002,0.164±0.012,0.294±0.031,0.473±0.008,0.605±0.005,0.583±0.007。与对照组相比，GILZ过表达不能显著促进前脂肪细胞的增殖(F=2.460,P=0.068),提示GILZ对脂肪细胞的增殖没有明显影响。2. GILZ-shRNA/pLentiLox 3.7测序结果与设计序列一致,将包装的shRNA慢病毒感染至3T3-L1细胞,48h后荧光显微镜观察,可见95%以上细胞呈绿色。GILZ-shRNA慢病毒感染至3T3-L1细胞48h后,在GILZ稳定表达3T3-L1细胞内,以实时荧光定量PCR法检测GILZmRNA表达,与pLentiLox 3.7空载体对照组相比,shRNA慢病毒基因沉默组GILZmRNA表达显著降低(0.191±0.027 vs0.125±0.018,t=3.459,p=0.026)3.3T3-L1细胞生长融合48h后,用脂肪细胞诱导基MID处理48h,然后换成10μg/ml胰岛素的正常培养基(隔天换液)培养9天,油红0染色显示,与对照组相比,GILZ过表达组的桔红色细胞大大的减少。用100%异丙醇溶解油红0染色的细胞,500nm吸光波长测定分化9天的脂肪细胞内甘油三酯相对含量显著降低(0.365±0.012 vs 0.181±0.014,F=80.023,P=0.000)。以GILZ-shRNA沉默GILZ表达能减少这种抑制作用,与对照组相比,甘油三酯相对含量显著增加(0.184±0.027 vs 0.335±0.029,F=27.189,P=0.000)。4.MID诱导后,实时荧光定量PCR法检测显示,分化过程中GILZ过表达的3T3-L1细胞内脂肪细胞分化基因PPARγ2, C/EBPα, LPL和FAS的mRNA表达显著降低(分化第九天时分别为11.447±0.831 vs 1.173±0.290,17.700±0.915 vs1.557±0.384,67.057±5.288 vs 9.467±3.406,40.946±3.968 vs 4.967±1.091,P=0.000)；而沉默GILZ表达却能显著增加这些基因的表达(1.056±0.275 vs6.162±0.432,1.500±0.644 vs 9.147±0.958,8.967±1.046 vs 45.127±2.585,4.518±1.143 vs 27.974±1.634,P=0.000)。小结：1. GILZ对前脂肪细胞的增殖没有明显的影响。2.制备的shRNA慢病毒能成功沉默GILZ的mRNA表达。3. GILZ过表达或沉默可显著性抑制或增加PPARγ2, C/EBPα, LPL和FAS的mRNA表达,表明GILZ可能通过下调脂肪细胞分化转录因子PPARγ2, C/EBPα的表达而抑制脂肪细胞特异性基因LPL和FAS的表达,进而抑制脂肪细胞的分化。4. GILZ可抑制C/EBPα的表达。第三部分：C/EBP和AP-1顺式作用元件在GILZ抑制PPARγ2启动子转录活性中的作用目的：通过分析C/EBP和AP-1顺式作用元件在GILZ抑制PPARγ2启动子转录活性的作用,探讨GILZ抑制脂肪细胞的调控机制。方法：使用荧光素酶(Luciferase)双报告基因系统检测GILZ对PPARγ2启动子区各顺式作用元件转录活性的影响。1.分别用0.1μg-615/-265-Luc报告基因(涵盖了PPARγ2启动子区的含C/EBP顺式作用元件)、-265/-1-Luc报告基因(涵盖了PPARγ2启动子区的含AP-1顺式作用元件),同时转染0.01μg pRL-SV40(起校正作用报告基因)、PcDNA3(对照)与HA-GILZ/PcDNA3不同比例的质粒于3T3-L1细胞,48h后检测荧光素酶活性。根据PcDNA3与HA-GILZ/PcDNA3不同比例,共分四组：1,对照组,0.45μg PcDNA3；2,0.30μg PcDNA3+0.15μg HA-GILZ/PcDNA3；3,0.15μg PcDNA3+0.30μg HA-GILZ/PcDNA3；4,0.45μg HA-GILZ/PcDNA3。2.分别用0.1μg-615/-1-Luc(含C/EBP和AP-1的PPARγ2启动子区报告基因)、-615/-265-Luc、-265/-1-Luc,同时转染0.01μg pRL-SV40、PcDNA3或0.45μg HA-GILZ/PcDNA3质粒于3T3-L1细胞,48h后检测荧光素酶活性。根据报告基因的不同,共分四组：1,对照组,0.45μg PcDNA3+0.1μg-615/-1-Luc；2, 0.45μg HA-GILZ/PcDNA3+0.1μg -615/-265-Luc；3, 0.45μg HA-GILZ/PcDNA3+0.1μg-265/-1-Luc；4,0.45μg HA-GILZ/PcDNA3+0.1μg-615/-1-Luc结果：1.Luciferce检测显示HA-GILZ可以显著性降低含C/EBP顺式作用元件的-615/-265-Luc报告基因转录活性(46.353±3.267 vs 22.683±1.588,F=44.394,P=0.000),随着GILZ量的增加而抑制作用增加。2.Luciferce检测显示HA-GILZ可以显著性降低含AP-1顺式作用元件的-265/-1-Luc报告基因转录活性(46.476±1.859 vs 29.039±1.639,F=18.293,P=0.001),随着GILZ量的增加而抑制作用增加。3.Luciferce检测显示HA-GILZ对含C/EBP顺式作用元件的PPARγ2启动子转录活性的抑制作用强于含AP-1顺式作用元件的PPARγ2启动子转录活性(22.567±1.653 vs 29.121±0.934,F=211.57,P<0.05),而含C/EBP和AP-1顺式作用元件的PPARγ2启动子-615/-1-Luc转录活性比单独的C/EBP或AP-1顺式作用元件则更显著性降低(14.157±0.527,P<0.05)。小结：1.C/EBP和AP-1顺式作用元件二者在GILZ对PPARγ2启动子转录活性的抑制作用中都起了明显的作用。2. C/EBP和AP-l顺式作用元件在G工LZ抑制PPARγ2启动子转录作用中,C/EBP作用要强于AP-1。3. C/EBP和AP-1顺式作用元件在GILZ抑制PPARγ2启动子转录作用中,具有协同作用。结论：三部分实验总结如下：1.成功构建HA-GILZ/PcDNA3重组表达载体和建立GILZ稳定表达的3T3-L1前脂肪细胞分化模型。2.免疫荧光显示GILZ在C3H10T1／2和3T3-L1细胞的细胞质和细胞核均有表达,但主要表达于细胞质。3.实验表明GILZ对前脂肪细胞的增殖没有明显的影响,但可抑制3T3-L1前脂肪细胞的分化。这种抑制作用的机制之一可能是GILZ通过PPARγ2启动子上的C/EBP、AP-1元件参与抑制PPARγ2启动子转录,从而进一步抑制了脂肪细胞分化相关基因PPARγ2,C/EBPα,LPL和FAS的mRNA表达。更多还原

【Abstract】 Adipocytes is a type of functional mammalian cells which play a key role in maintaining lipid and energy metabolism balance. Adipocytes differentiation is a very complex process which many transcription factors,cell factors and hormones involves in regulating it from pre-adipocytes to mature adipocytes.Dysfunction of adipocytes differentiation may lead to many diseases, such as obesity, type 2 diabetes mellitus, fatty liver,hyperlipidemia and mammary cancer,et al.Investigation of pre-adipocytes proliferation and differentiation is the core of obesities mechanism,and it is beneficial to look for the drug of body weight reduction. So, the research on adipocytes differentiation in molecular and cellular levels has a theoretical and practical significance on studing the above life and diseases process,as well as curing and preventing these diseases.Glucocorticoid (GC) induced leucine zipper (GILZ) has been identified as a glucocorticoids-induced protein, has many important biological functions, such as inhibiting inflammation and protecting T cells from apoptosis et al. GC regulates a spectrum of cell functions, including growth, differentiation, and apoptosis. It is also among the best known anti-inflammatory and immune suppressive agents and is widely used for treating diseases such as rheumatoid arthritis, pulmonary disease, asthma and in organ transplantation to prevent rejection. Importantly, long term GC treatment can accelerate bone loss and result in osteoporosis, as well as replace of marrow cell populations with adipose tissues (fatty marrow). Primary reports showed that GILZ antagonizes GC’s function by inhibiting GC induced adipogenesis and suggested that GILZ functions as an antagonist in the GC negative-feedback circuit. GILZ belongs to the leucine-zipper family of transcription factors.Several studies suggested GILZ as a transcriptional repressor to inhibit gene expression through direct association with nuclear factor-KB and activating protein. It also was found to involve in signal transduction of Fas/FasL、NF-κB、Ap1、Raf21 signal passways.During Adipocytes come into being, The peroxisome proliferator-activated receptor gamma (PPARy) family and C/EBPs play an important role as the target transcription factors in relative cell signal passways. PPARy belongs to the nuclear hormone receptor subfamily of transcription factors that can bind to specific DNA response elements in the regulatory regions of target genes and there are three protein products, PPARγ1, PPARγ2 and PPARγ3, which are isoforms transcribed from the same gene with different promoter usage.PPARy2 is predominantly expressed in adipose tissue. Of these three isoforms, PPARy2 is a key regulator of adipogenesis and is expressed at an early stage of the adipogenesis program. PPARy2 can activate a battery of genes necessary for lipid metabolism, including lipoprotein lipase (LPL), phosphoenolpyruvate carboxykinase, fatty acid-binding and transport proteins, and stearoyl-CoA desaturase-1 (SCD-1). Functional PPAR response elements (PPREs) have been identified in the promoter regions of these genes. Another important transcription factor family of adipocytes differentiation C/EBPs, which have three protein isoforms, C/EBPa, C/EBPβand c/EBPδ. All of them can promote adipocytes differentiation. C/EBPa maybe mediates the effects of GILZ on adipocytes differentiation and it can active transcription with PPARy2 each other to further initiate adipocytes differentiation.GILZ acts as an important kind of signal molecule and nuclear transcription factor, However, it is still unclear whether GILZ involves in adipocytes differentiation, what about its regulatory mechanisms,and whether C/EBPaand PPARy2 mediate the effect of GILZ. Therefore,To elucidate the effects of GILZ on adipocytes differentiation and its regulatory mechanism has an important signality for curing and prevention of obesity, type-2 diabetes mellitus.This thesis is divided into three parts to investigate the effect of GILZ on adipocytes differentiation and its regulatory mechanisms:1)Generally construct HA-GILZ/PcDNA3 recombinant mammalian expression vector and identify its expression and location in cell lines.3T3-L1 preadipocytes differentiation is induced with obutylmethylxanthine, insulin and dexamethasone,and set up the cell model of adipocytes differentiation. After 3T3-L1 cells were transfected with Lipofeactamine 2000 liposome and screened with G418, HA-GILZ stable and high expressed cell lines is cloned. It mainly aimed to provide gene and cell tools for studying the biology functions of GILZ.2)Compare the effect of GILZ on preadipocytes proliferation, special color of lipid in differentiation adipocytes and the relative mark gene PPARγ2, C/EBPα, LPL and FAS mRNA expression. Study the biology functions of GILZ and its regulatory mechanism at cell and mRNA expression.3)Primary analysis of the effect C/EBP and AP-1 cis-elements on GILZ inhibiting PPARy2 promotor transcript activities.Study the effect mechanisms of GILZ in molecular regulatory level of DNA transcription cis-element.Part 1:Construction of HA-GILZ mammalian expression vector and identified its expression and location in the cell lines, cloning GILZ stable expressed 3T3-L1 cell lines AIM:To provide the tools for studying the function and location of glucocorticoid induced leucine zipper (GILZ) in adipocytes differentiation, we constructed HA-GILZ expressive vector and observe the expression and location of HA-GILZ in cell lines, set up the differentiation cells and GILZ high stable expressed 3T3-L1 cell line models.METHODS:1.GILZ cDNA codon domain was amplified by RT-PCR from C3H10T1/2 cell line and recombined into PcDNA3 plasmid which was signed with HA-Tag. The recombinant vector HA-GILZ/PcDNA3 was identified by the assay of two restrictional enzymes BamH I/Xho I and sequencing.2.The recombinant vector HA-GILZ/PcDNA3 was instantaneously transfected into C3H10T1/2 cells by Lipofeactamine 2000 liposome and instantaneously transfected into 293T cells.3.The recombinant vector HA-GILZ/PcDNA3 was transfected into 3T3-L1 cells by Lipofeactamine 2000 liposome and the stable expressed cells were screened with G418.4.After 48h transfection, expression and location of GILZ in C3H10T1/2 and 3T3-L1 cells were detected with immunofluorescence,expression and molecular weight of GILZ in 293T cells and HA-GILZ expressed 3T3-L1 cells determinated with western bolt.5.3T3-L1 cell differentiation was induced with adipocyte induction medium (MID) containing isobutylmethylxanthine (0.5mM), insulin (10μg/ml) and dexamethasone (0.25μM). Lipid special staining and relative content of triglyceride were determined by Oil-O staining.RESULTS:1.Constructed recombinant vector HA-GILZ/PcDNA3 was confirmed successfully with the identifications of restrictional enzyme assay and sequencing analysis. Immunofluorescence further showed HA-GILZ fusion protein expressed in both cytoplasm and nuclear, however, it mainly expressed in the cytoplasm of cells. Western blot showed high-efficiently expressed in 3T3-L1 and 293T cell lines treansfected with recombinant vector HA-GILZ/PcDNA3 and has a special band of protein molecular weight about 20kD.2.After 3T3-L1 pre-adipocytes were induced 3 days with MID, some small lipid droplets were observed in cells and more and more cells appeared lipid droplets with the differentiation development. On the 9th days with MID, lots of round lipid droplets were seen in adipocytes cytoplasm, while cells grown in normal growth media had no lipid accumulation. Oil-Red O staining showed the lipid droplets in differentiation adipocyte is orange-red color. Compare with uninduced control, the relative content of triglyceride significantly increase (0.139±0.022 vs 0.356±0.014, t=14.521, p=0.000) in induced cells.3.Transfected 3T3-L1 cells were transfected with Lipofeactamine 2000 liposome and screened by G418, immunofluorescence and western blot showed GILZ is high expressed. Look like C3H10T1/2 cells, GILZ protein was also observed to mainly expressed in the cytoplasm of 3T3-L1 cells.CONCLUSIONS:1.HA-GILZ fusion gene vector was successfully constructed and the protein expressed in cytoplasm of C3H10T1/2 cells. It provides a stable gene tool for investigating the biologic function of GILZ.2.GILZ protein was observed to mainly express in the cytoplasm of C3H10T1/2 and 3T3-L1 cells.3.3T3-L1 pre-adipocytes differentiation was successfully induced and 3T3-L1 cell lines of stable expressed of GILZ was set up. It provides a stable cell tool for investigating the biologic function of GILZ.Part 2:The effect of GILZ on preadipocytes proliferation and adipocytes differentiation and relative gene expressions.AIM:To observe the effect of GILZ on 3T3-L1 pre-adipocytes proliferation, study the effects on mRNA expression of the relative marker genes PPARy2, C/EBPα,LPL and FAS of adipocytes differentiation and investigate the adipocyte differentiation mechanism of regulation of GILZ.METHODS:1. From day 1 to day 11, cell proliferation of 3T3-L1 cell clones of stable expression of GILZ was determinated every two days with MTT method.2.The effect of GILZ on adipocyte differentiation was analyzed with Oil-0 staining. It was divided into four groups:1, cells transfected with PcDNA3, un-induced with MID; 2, cells transfected with HA-GILZ/PcDNA3, un-induced with MID; 3, cells transfected with PcDNA3, induced with MID for 9 days; 4, cells transfected with HA-GILZ/PcDNA3, induced with MID for 9 days.3.Generally constructed GILZ-shRNA/pLentiLox 3.7 (short for GILZ-shRNA) recombinant Lentivirus vector, sequenced, transfected with Lipofeactamine 2000 liposome, coated with 293T cells, infected into 3T3-L1 cell clones of stable expression of GILZ, GILZ mRNA expression examined with Real-time PCR method.4.The mRNA expression of the relative marker genes PPARy2, C/EBPa, LPL and FAS of adipocytes differentiation were measured with Real-time PCR. For GILZ overexpression, it was divided into two groups:transfected PcDNA3 or HA-GILZ/PcDNA3; For GILZ silence, it was also divided into two groups:infected pLentiLox 3.7 and GILZ-shRNA/pLentiLox 3.7 reapectively.RESULTS:1. MTT method showed that, for 3T3-L1 control cells proliferation at the day lst,3rd,5th,7th,9th and day 11th, the ODs value were 0.090±0.008,0.154±0.004, 0.273±0.017,0.471±0.008,0.609±0.013,0.610±0.018 respectively; while for 3T3-L1 cells of stable expression of GILZ, the ODs value 0.083±0.002, 0.164±0.012,0.294±0.031,0.473±0.008,0.605±0.005,0.583±0.007respectively. Compared with control, GILZ overexpression can not obviously promote pre-adipocytes proliferation (F=2.460, P=0.068). That is to say, GILZ has no obviously effect on adipocytes proliferation.2.The sequencing result of GILZ-shRNA/pLentiLox 3.7 was consisting with design. After 3T3-L1 cells were infected with coated GILZ-shRNA for 48h, cells more than 95% were observed green under fluorescent microscopy. After 48h, Real-time PCR detected GILZ mRNA expression in 3T3-L1 cells of stable expression of GILZ. Compared with the control, GILZ mRNA expression significantly decreased in GILZ-shRNA Lentivirus silence group (0.191±0.027 vs 0.125±0.018, t=3.459, p=0.026) 3.After cell confluent for 48h,3T3-L1 cells were treated with MID for 48h, then changed into culture medium containing 10μg/ml insulin (the other day changed medium) for 9 days. Oil-Red O staining showed, compared with the control, orange-red cells in GILZ overexpression group greatly decreased. The Oil-Red O stained cells were dissolved with 100% isopropyl alcohol, and the relative content of triglyceride at the day 9th of adipocyte differentiation was measured by OD500. The relative content of triglyceride significantly also decreased (0.365±0.012 vs 0.181±0.014, F=80.023, P=0.000),while GILZ expression of SiRNA silence can decrease this inhibition. Compared with the control, the relative content of triglyceride significantly increased (0.184±0.027 vs 0.335±0.029, F=27.189, P=0.000).4.After induction with MID, Real-time PCR showed PPARγ2, C/EBPa,LPL and FAS mRNA expression significantly decreased (at cell differentiation 9 days, 11.447±0.831 vs 1.173±0.290,17.700±0.915 vs 1.557±0.384,67.057±5.288 vs 9.467±3.406,40.946±3.968 vs 4.967±1.091, P=0.000), while ShRNA silence can significantly increase these gene expression (1.056±0.275 vs 6.162±0.432, 1.500±0.644 vs 9.147±0.958,8.967±1.046 vs 45.127±2.585,4.518±1.143 vs 27.974±1.634, P=0.000).CONCLUSIONS:1.Our study indicated that GILZ has no effect on preadipocyte proliferation.2.Prepared GILZ-shRNA Lentivirus can successfully silence GILZ expression.3.GILZ overexpression or silence can significantly inhibits or increases PPARγ2, C/EBPa, LPL and FAS mRNA expression, which indicated that down-regulation of adipocytes differentiation transcript factors PPARy2 and C/EBPa inhibits adipocytes special gene LPL and FAS expression, so as to further inhibit adipocyte differentiation.4. GILZ can inhibit C/EBPa expression. Part 3:The effect of C/EBP and AP-1 cis-elements on GILZ inhibiting PPARy2 promotor transcript activities.AIM:To further study the regulatory mechanism of GILZ inhibiting adipocytes differentiation, the effect of C/EBP and AP-1 cis-elements on GILZ inhibiting PPARy2 promotor transcript activities were analyzed.METHODS:The effect of C/EBP and AP-1 cis-elements on GILZ inhibiting PPARy2 promotor transcript activities was quantified with luciferase dual reporter gene.1. After 3T3-L1 cells were transfected with 0.1μg-615/-265-Luc reporter gene (containing C/EBP cis-element of PPARy2 promotor region) or-265/-1-Luc reporter gene (containing AP-1 cis-element of PPARy2 promotor region), together with 0.01μg pRL-SV40 (reporter gene ascalibration), PcDNA3 (contorl) and HA-GILZ/PcDNA3 plasmid in different proportion for 48h, luciferase activities were determinated. Based on the different proportion of PcDNA3and HA-GILZ/PcDNA3 plasmid, it is divided into four groups:1, contorl,0.45 [μg PcDNA3; 2,0.30μg PcDNA3+0.15μg HA-GILZ/PcDNA3; 3,0.15μg PcDNA3+0.30μg HA-GILZ/PcDNA3; 4,0.45μgHA-GILZ/PcDNA3.2. After 3T3-L1 cells were transfected with 0.1μg-615/-1-Luc reporter gene (containing both of C/EBP and AP-1 cis-elements of PPARy2 promotor region),-615/-265-Luc reporter gene,-265/-1-Luc reporter gene respectively, together with 0.01μg pRL-SV40 (reporter gene ascalibration), PcDNA3 (contorl) and HA-GILZ/PcDNA3 plasmid in different proportion for 48h, luciferase activities were determinated. Based on the different reporter genes, it is divided into four groups:1, contorl,0.45μg PcDNA3+0.1μ-615/-1-Luc; 2,0.45μg HA-GILZ/PcDNA3+0.1μg-615/-265-Luc; 3,0.45μg HA-GILZ/PcDNA3+0.1μg-265/-1-Luc; 4,0.45μg HA-GILZ/PcDNA3+0. 1μg-615/-1-Luc.RESULTS: 1. Luciferase assay showed that GILZ can significantly decreased transcript activities of -615/-265-Luc reporter gene containing C/EBP cis-element (46.353±3.267 vs 22.68±1.59, F=44.394, P=0.000)and the inhibiting effect enhenced along with GILZ content increased.2. Luciferase assay showed that GILZ can significantly decreased transcript activities of-265/-1-Luc reporter gene containing AP-1 cis-element (46.476±1.859 vs 29.039±1.639, F=18.293, P=0.001) and the inhibiting effect enhenced along with GILZ content increased.3. Luciferase assay showed that the inhibitory effect of HA-GILZ on the transcript activitie of reporter gene containing C/EBP cis-element is significantly stronger than that of containing AP-1 cis-element (22.567±1.653 vs 29.121±0.934, F=211.567, P＜0.05). The transcript activitie of-615/-1-Luc reporter gene containing both C/EBP and AP-1 cis-elements is significantly lower than those of containing separate C/EBP or AP-1 cis-element (14.157±0.527, P＜0.05)CONCLUSIONS:1. Both C/EBP and AP-1 cis-elements GILZ play obvious effect on GILZ inhibiting PPARy2 promotor transcript activities.2. When C/EBP and AP-1 cis-elements involved in the inhibtory effects of GILZ on transcript activities of PPARy2 promotor, the inhibitory effect of C/EBP cis-element is higher than that of AP-1.3. When C/EBP and AP-1 cis-elements involved in the inhibtory effects of GILZ on transcript activitie of PPARy2 promotor, they can cooperate each other.SUMMARY:We summarized the three-part tests as following:1. Successfully constructed recombinant vector of HA-GILZ/PcDNA3 and set up 3T3-L1 cell differentiation model of stable expression of GILZ.2.Immunofluorescence showed GILZ protein expressed in both cytoplasm and nuclear of C3H10T1/2 and 3T3-L1 cells. However, it mainly expressed in the cytoplasm of cells.3.Our study indicated that GILZ has no effect on preadipocyte proliferation, but it can inhibit 3T3-L1 pre-adipocyte differentiation. We suggested that one of the probably inbiting mechanisms is down-regulation of adipocytes differentiation relative genes PPARy2, C/EBPa, LPL and FAS expression via C/EBP and AP-1 cis-element of PPARy2 promotor region involving in GILZ inhibiting PPARy2 transcription.更多还原