【作者】 狄文娟；

【导师】 丁国宪；

【作者基本信息】 南京医科大学 ， 老年医学， 2009， 硕士

【摘要】 目的:目前肥胖已成为全球流行性疾病,而中心性肥胖与2型糖尿病密切相关。过氧化物酶体增殖物激活受体γ(PPARγ)与类固醇受体均为核受体家族成员,且都参与脂肪代谢的相关基因的转录调控。胰岛素增敏剂——噻唑烷二酮类是PPARγ的选择性激动剂,在临床使用中,其表现出体重增加的副作用,而这副作用包括促进脂肪再分布及水钠潴留。而众所周知,糖皮质激素可以导致向心性肥胖,且可以减低胰岛素敏感性,增加血糖水平。结合PPARγ与类固醇受体均有促进脂肪聚积的功能,本研究用TZDs——吡格列酮对基因肥胖小鼠及3T3-L1前脂肪细胞进行药物干预和刺激,分别从体内和体外两方面来研究PPARγ与类固醇受体在肥胖中的作用及相互之间的影响。同时也为进一步研究TZDs的副作用提供研究基础。方法:1.将ob/ob小鼠分为正常对照组、吡格列酮短期治疗组及长期治疗组。对照组给予安慰剂、治疗组分别予吡格列酮皮下注射2周或6周,观察用药后小鼠体重,脂肪分布变化;利用实时荧光定量PCR技术检测脂肪组织及肾脏中的MR、GR及其相关基因mRNA的表达量。2. 3T3-L1脂肪前体细胞生长接触至单层后,采用3-异丁基-1-甲基黄嘌呤、地塞米松及胰岛素的联合诱导方案诱导其分化至14天,同时以PPARγ激动剂——吡格列酮对分化过程中的细胞进行刺激,采用油红O染色,在倒置显微镜下观察细胞形态、脂滴的形成,以及通过异丙醇萃取油红O的方法对脂滴进行定量;采用实时荧光定量PCR技术观察脂肪细胞分化过程中MR、GR及其相关基因mRNA的表达量的变化,以及给予吡格列酮刺激后对三者表达的影响。结果:1.与正常ob/ob小鼠组比较,吡格列酮给药组体重逐渐增加,且均有统计学差异(P<0.05);吡格列酮短期给药组小鼠皮下及内脏脂肪重量较正常组无统计学意义,长期给药组皮下脂肪明显较正常组增加(P<0.01),附睾旁脂肪仅有增长趋势,无统计学差异;皮下脂肪中,吡格列酮治疗组MR、GR及11β- HSD1的mRNA表达的有明显增长;内脏脂肪MR、GR及11β- HSD1的mRNA含量无统计学变化;肾脏组织中,短期给药后11β- HSD2、MR及GR的mRNA表达量明显降低(P<0.05),但长期给药后11β- HSD2、MR较短期组不同程度增高。2.随着诱导时间的增加3T3-L1细胞明显分化,分别对第4、8、14天收取细胞进行油红O染色及异丙醇萃取定量,吡格列酮组的分化程度(约90%)明显高于正常诱导组(约60%)。用荧光实时定量PCR对该细胞的MR、GR及11β- HSD1的mRNA的含量进行检测,结果表明其表达随着脂肪细胞的成熟明显上调(P<0.01)。在3T3-L1前脂肪细胞分化过程中,吡格列酮刺激组MR、GR及11β- HSD1的mRNA表达量均较正常组增加,在第14天与正常诱导组比较,显著高于正常组(P<0.01)。结论:1. PPARγ激动剂可以促使ob/ob小鼠皮下脂肪增多及MR、GR及11β- HSD1的mRNA表达量增加,而对内脏脂肪无明显影响。说明了PPARγ激动剂的皮下脂肪在再分布作用可能与类固醇受体相关。2.吡格列酮对3T3-L1前脂肪细胞分化过程持续刺激后,发现吡格列酮能明显促使3T3-L1前脂肪细胞分化率增加;前脂肪细胞中表达盐皮质激素,其表达与细胞的成熟度正相关;吡格列酮在促进前脂肪细胞的分化的同时,明显增加GR, MR及11β-HSD1的表达,从细胞机制说明了吡格列酮增加肥胖的副作用与类固醇激素受体的相互作用。更多还原

【Abstract】 Objective:Obesity, characterized by excess adipose tissue, is a common health problem with increasing prevalence around the world. Obesity, particularly central obesity, is an important risk factor for type 2 diabetes and cardiovascular disease. Peroxisome-proliferator–activated receptorγ(PPARγ) and steroid receptors are nuclear receptors, which regulate transcriptional responses to diverse signaling pathways in adipose tissues. The insulin-sensitizing thiazolidinediones, which are selective ligands of PPARγ, have a side effect of body weight gain. It is attributed to expansion of the subcutaneous fat depot, and in some patients to edema, whereas the mass of visceral fat remains unchanged or decreases. Glucocorticoids, the ligand of glucocorticoid receptor and mineralocorticoid receptor induce fat redistribution and accumulation. And fat is shed from limbs and accumulates in truncal and visceral areas. To explore the relationship between PPARγand steroid receptors, we investigated the mechanisms of thiazolidinediones’adverse effects by using the ob/ob mice model and 33T3-L1 preadipocytes, and compared the different effects in adipose tissue with or without pioglitazone.Methods:1. After having balanced for 3d, the ob/ob mice were randomly divided into the normal group and pioglitazone-treatment group. And they were injected hypodermically with placebo or PGZ for two or six weeks. The weight and adipocyte distribution were observed. The mRNA expression levels MR, GR and the related gene from adipose tissue were analyzed by real-time quantitative PCR.2. Postconfluent 3T3-L1 preadipocytes were incubated with a cocktail of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX) in DMEM supplemented with 10% fetal calf serum (FCS) for 48 h, with the culture medium replaced next 48 h with DMEM, supplemented with 10% FCS and insulin, and then refeeding with standard medium. 3T3-L1 preadipocytes were treated with or without pioglitazone during differentiation. We observed the adipocytes on day0, day4, day8 and day14 under microscope, with the cells stained by oil red O. And real-time quantitative PCR was used to study the mRNA expression levels of MR, GR and the related gene.Results:1. The time-depended weight(P<0.05)were raised in PGZ-treatment group. The mass of subcutaneous adipose were raised in long-time treatment-group(P<0.01), and there were no statistical significance between the mass of visceral adipose tissues in two groups. Comparing to control group, the mRNA levels of MR, GR and 11β- HSD1 in PGZ groups were significantly raised in subcutaneous adipose tissues, and no change in visceral adipose tissues. In kidney, with the treatment of PGZ, the mRNA expressions of MR and 11β- HSD2 upregulated.2. With the process of adipose differentiation under classical conditions, 3T3-L1 fibroblasts were long-term exposure to 10μM pioglitazone, an agonist of PPARγ. After differentiation, the 3T3-L1 preadipocytes turned to be larger and round rather than of spindle shape, and contained large droplets of triglycerides. The cells were stained in day4, day8 and day 14 during the differentiation by oil red O, and quantitated by isopropyl alcohol method. Over 60% of the preadipocytes in control group and about 90% of the pioglitazone treatmented cells appeared to be differentiated into mature adipocyte. However, the single cell in PGZ group was smaller than control group’s. The mRNA levels of MR and relative genes were detected by real-time quantitative PCR. Accompanied with pioglitazone induced adipogenesis, mRNA levels of MR and 11β-hydroxysteroid dehydrogenase type 1 were increased(P<0.01).Conclusion:3. PPARγagonist–pioglitazone could increase the weight of ob/ob mice, and improve the expansion of the subcutaneous fat depot, with the raise of the mRNA levels of steroid receptors. This study proved the relationship between side effects of pioglitazone and steroid receptors in vivo.4. Pioglitazone promote the differentiation of 3T3-L1 preadipocytes. 3T3-L1 preadipocytes express the mRNA of steroid receptors, which were increased during differentiation. Accompanied with pioglitazone induced adipogenesis, the mRNA levels of steroid receptors were increased. It is conclude that pioglitazone have the effect on steroid receptors.更多还原