【作者】 姜友昭；

【导师】 陈兵；

【作者基本信息】 第三军医大学 ， 内科学， 2007， 博士

【摘要】 背景和目的PPAR是一组核受体超家族成员,有PPARα、δ或β、γ三种亚型。目前关于PPARα和PPARγ的结构和功能基本上已经明确。但是PPARδ作为最后鉴定的PPAR,其功能目前尚无深入研究。研究发现,PPARδ分布相当广泛,在胰岛、心肌、骨骼肌、脂肪组织、皮肤、脑组织中均有较高表达。研究还发现在骨骼肌、心肌、脂肪组织中,PPARδ均能够增强脂肪酸的氧化和利用,是脂肪酸氧化酶的上游调节因子。因此PPARδ被看作代谢综合征的新的强有力的治疗靶点。但是也有研究发现,在巨噬细胞中PPARδ能够促进胆固醇内流和脂质沉积。这些研究提示PPARδ在脂质代谢中的作用可能具有组织特异性。脂毒性导致的胰岛β细胞功能障碍是代谢综合征发病的中心环节之一,改善胰岛β细胞脂代谢可能对代谢综合征的防治起着重要作用。虽然目前有较多的提示性证据表明PPARδ可能在调节胰岛β细胞脂代谢和胰岛素分泌中起作用,但是其在胰岛β细胞中的确切功能却无直接的实验研究报道。为此我们采用Adeasy系统,构建了PPARδ、PPARδ显性负性突变体PPARδ-DN、PPARδ-shRNA的腺病毒载体,观察了它们对INS-1细胞脂代谢相关酶的表达、细胞内甘油三酯浓度、胰岛素释放及相关基因表达的影响,以期明确PPARδ对胰岛β细胞脂代谢和胰岛素分泌的影响及初步机制。方法1.在葡萄糖浓度分别为3 mmol/L、11 mmol/L、20 mmol/L培养条件下,分别用RT-PCR和western blot检测PPARδmRNA和蛋白的表达变化。2.在软脂酸浓度分别为0.125 mmol/L、0.25 mmol/L、0.5 mmol/L培养条件下,分别用RT-PCR和western blot检测PPARδmRNA和蛋白的表达变化。3.采用Adeasy系统,构建PPARδ和PPARδ的显性负性突变体PPARδ-DN的重组腺病毒载体。4.在构建了PPARδsiRNA表达质粒的基础上,采用Adeasy系统构建PPARδ-shRNA的重组腺病毒载体。5.分别用PPARδ腺病毒、PPARδ-DN腺病毒、PPARδ-shRNA腺病毒感染INS-1细胞,RT-PCR检测其对INS-1细胞脂代谢相关基因ACO、CPT1、FATP1、LCAD表达的影响。同时检测细胞内甘油三酯浓度;6.分别用PPARδ腺病毒、PPARδ-DN腺病毒、PPARδshRNA腺病毒感染INS-1细胞,RT-PCR检测它们对INS-1细胞Preproinsulin基因表达和放免法检测它们对INS-1细胞胰岛素释放的影响。7.分别用PPARδ腺病毒、PPARδ-DN腺病毒、PPARδshRNA腺病毒感染INS-1细胞,RT-PCR分别检测它们对GLUT2、PDK4、UCP-2 mRNA表达的影响。结果1. INS-1细胞的PPARδmRNA及蛋白表达随着葡萄糖浓度的升高而逐渐降低;2. INS-1细胞的PPARδmRNA及蛋白表达随着软脂酸浓度的升高而逐渐升高。3.成功构建了PPARδ和PPARδ的显性负性突变体PPARδ-DN的腺病毒载体。4.成功构建了PPARδ的siRNA表达载体,并在此基础上,成功构建了PPARδ-shRNA腺病毒表达载体。5. PPARδ腺病毒感染INS-1细胞后,INS-1细胞脂代谢相关基因ACO、CPT1、FATP1、LCAD的表达明显升高,细胞内甘油三酯含量明显降低。而PPARδ-DN腺病毒、PPARδ-shRNA腺病毒感染INS-1细胞后,ACO、CPT1、FATP1、LCAD的表达明显降低,细胞内甘油三酯含量明显升高。6. PPARδ腺病毒感染INS-1细胞后,INS-1细胞培养基中胰岛素含量明显减少,Preproinsulin表达明显下降。而PPARδ-DN腺病毒、PPARδ-shRNA腺病毒感染INS-1细胞后,INS-1细胞培养基中胰岛素含量明显上升,Preproinsulin表达明显增高。7. PPARδ腺病毒感染INS-1细胞后,UCP-2、PDK4的表达明显升高, GLUT2的表达明显下降。而PPARδ-DN腺病毒、PPARδshRNA腺病毒感染INS-1细胞后,UCP-2、PDK4的表达明显下降,而GLUT2的表达明显增高。结论1. INS-1细胞中,葡萄糖是PPARδ表达的负向调控因素,而脂肪酸是PPARδ表达的正向调控因素。提示PPARδ在胰岛β细胞的糖脂代谢中起着重要作用。2. PPARδ能够促进胰岛β细胞的脂肪酸氧化,减少胰岛β细胞内的甘油三酯沉积,从而可能减轻胰岛β细胞的脂毒性。3. PPARδ能够抑制胰岛β细胞的胰岛素分泌。PPARδ抑制胰岛β细胞胰岛素分泌的机制可能包括增强UCP-2、PDK4和下调GLUT2的表达。4. PPARδ对胰岛功能的影响可能是多方面的:首先,PPARδ促进胰岛β细胞的脂肪酸氧化,减少胰岛β细胞内的甘油三酯沉积,从而可能减少胰岛β细胞的脂性凋亡;其次,PPARδ能够降低胰岛素分泌从而可能防止长期高脂条件下的胰岛β细胞功能耗竭。最后,在短期内,PPARδ抑制胰岛β细胞胰岛素分泌,这也可能加重胰岛β细胞的糖毒性。因此,PPARδ对胰岛β细胞功能的影响需要进一步的长期的观察。更多还原

【Abstract】 Background and ObjectivePPAR, which contains three isotypes of PPARα, PPARδ(β) and PPARγ, belongs to the nuclear receptor superfamily. By now, the structure and function of PPARαand PPARγare clear, but as the lastly identified isotype, the function of PPARδremains obscure. PPARδis widly expressed in the islet, cardiac muscle, skeletal muscle, adipose tissue, skin and brain. In the cardiac muscle, skeletal muscle and adipose tissue, PPARδis an important regulator of fatty acid oxidation. The up-regulation of PPARδor activation of PPARδby its agonists could enhance the oxidation and utilization of fatty acids, so PPARδis believed to be the upper regulator of fatty acids oxidase and a potential target for the treatment of metabolic syndrome. By contrast, some specialist reported that PPARδpromoted cholesterol influx and lipid accumulation in macrophage, which is opposite to the result in other tissues. These results indicate that the role of PPARδin lipid metabolism may be tissue-specific.Impairment of isletβcells by 1ipotoxicity plays a central role during the development of type 2 diabetes mellitus, and the improvement of lipid metabolism may contribute to the prevention and treatment of metabolic syndrom. By now, there are much indirect data shows that PPARδmay play an important role in the regulation of lipid metabolism and insulin release inβcells. But we don’t find any direct report of the function of PPARδinβcells. So we adopt the Adeasy system to get the adenovirus vector of PPARδ, the dominant negative mutant of PPARδ(PPARδ-DN) and PPARδ-shRNA, by which we could observe the effect of PPARδon the expression of fatty acids oxidase, TG concentration, insulin content and insulin-related genes. Finally we could get the effect of PPARδon lipid metabolism, insulin release of pancreaticβcells and their elementary mechanism.Methods1. The INS-1 cells were incubated in the media containing 3 mmol/L, 11 mmol/L and 20 mmol/L glucose for 24 hours, respectively, then the PPARδmRNA was detected by RT-PCR and the PPARδprotein level was determined by Western blot.2. The INS-1 cells were incubated in the media containing 0.125 mmol/L, 0.25 mmol/L and 0.5 mmol/L PA for 24 hours, respectively, then the PPARδmRNA was detected by RT-PCR and PPARδprotein level was determined by Western blot.3. The Adeasy system was used to construct the PPARδand PPARδdominant negative mutant (PPARδ-DN) adenovirus vector.4. The PPARδ-shRNA adenovirus vector was constructed after the PPARδsiRNA plasmid pGensil/PPARδwas constructed successfully.5. The INS-1 cells were infected by PPARδadenovirus, PPARδ-DN adenovirus and PPARδ-shRNA adenovirus, then the mRNA level of ACO, CPT1, FATP1 and LCAD was detected by RT-PCR. At the same time, the intracellular content of TG was determined.6. The INS-1 cells was infected by PPARδadenovirus, PPARδ-DN adenovirus and PPARδ-shRNA adenovirus, then the mRNA level of preproinsulin was detected by RT-PCR and the insulin content was determined by RIA.7. The INS-1 cells was infected by PPARδadenovirus, PPARδ-DN adenovirus and PPARδ-shRNA adenovirus, then the mRNA level of GLUT2, PDK4 and UCP-2 was detected by RT-PCR.Results1. The expression of PPARδmRNA and protein were both down-regulated by glucose.2. The expression of PPARδmRNA and protein were both up-regulated by PA.3. The PPARδand PPARδ-DN adenovirus vectors were constructed successfully.4. The pGensil/PPARδ-shRNA was constructed successfully, and then the PPARδ-shRNA adenovirus vector was constructed.5. The mRNA level of ACO, CPT1, FATP1 and LCAD was higher in INS-1 cells infected by PPARδadenovirus than that in the controls. And the level of them was much lower in INS-1 cells infected by PPARδ-DN and PPARδ-shRNA adenovirus. By contrast, the TG content in INS-1 cells infected by PPARδwas much lower than the controls and opposite results were observed in INS-1 cells infected by PPARδ-DN and PPARδ-shRNA adenovirus.6. In INS-1 cells infected by PPARδadenovirus, the insulin content and the mRNA level of preproinsulin were lower than the contols. By contrast, the insulin content and the mRNA level of preproinsulin were much higher than the contols in INS-1 cells infected by PPARδ-DN and PPARδ-shRNA adenovirus.7. PPARδadenovirus upregulated the mRNA level of PDK4, UCP-2 and down-regulated the expression of GLUT2. But the PPARδ-DN and PPARδ-shRNA adenovirus down-regulated the mRNA level of PDK4, UCP-2 and up-regulated the expression of GLUT2.Conclusion1. The glucose is a negative regulator and fatty acids is a positive regulator of the PPARδexpression in INS-1 cells, which suggest that PPARδmay play an important role in lipid and glucose metabolism of pancreaticβcells.2. PPARδcould enhance the fatty acids oxidation and alleviate the lipid deposition in INS-1 cells, which contribute to lighten the 1ipotoxicity of INS-1 cells.3. PPARδcould depress insulin release of INS-1 cells, and its mechanism may involve the upregulation of PDK4、UCP-2 and down-regulation of GLUT2.4. The effect of PPARδon islet may be complicated: First, PPARδenhances the fatty acids oxidation and alleviates the lipid deposition, whichi may lighten the 1ipotoxicity-induced apoptosis. Second, PPARδabate the hyperinsulinemia in condition of hyperlipemia to prevent exhausting insulin release capacity. Third, the attenuation of insulin release by PPARδmay aggravate the glucotoxicity on islet. So the prolonged effect of PPARδonβcells need to be studied in the future.更多还原