【作者】 吴佩文；

【导师】 杨立勇；

【作者基本信息】 福建医科大学 ， 内科学， 2011， 博士

【摘要】 游离脂肪酸(FFAs)短期干预可促进β细胞分泌胰岛素,长期干预抑制β细胞的功能。研究发现FFAs受体G蛋白偶联受体40(GPR40)能介导胰岛素分泌,在2型糖尿病的病理生理机制中起重要作用。尽管进行了许多研究,目前GPR40的生物学功能还未完全清楚。为了对GPR40有更加深入的认识,本研究探讨GPR40是否介导FFAs对胰岛β细胞葡萄糖刺激的胰岛素分泌(GSIS)的短期和长期影响;是否介导FFAs对β细胞胰-十二指肠同源盒基因-1(PDX-1)和葡萄糖转运蛋白2(GLUT2)表达的影响,以及是否参与吡格列酮对胰岛β细胞脂毒性的干预作用。本研究第一部分构建稳定表达GPR40shRNA的βTC6细胞株。首先设计合成两对含GPR40shRNA的寡核苷酸模板链,连接入线性pSilencer4.1-CMV neo siRNA表达载体,并测序鉴定。脂质体法把载体转染至βTC6细胞,通过G418筛选得到稳定转染的βTC6细胞株。RT-PCR和western blot检测细胞GPR40 mRNA和蛋白的表达情况。结果显示成功构建了GPR40 shRNA重组质粒,转染重组质粒pSilencer-GPR40shRNA的βTC6细胞GPR40 mRNA和蛋白表达比对照组显著降低,建立了持续低表达GPR40的βTC6细胞株。本研究第二部分旨在探讨GPR40是否介导FFAs和吡格列酮作用下βTC6细胞的GSIS。分别以不同浓度FFAs(0.25、0.5或1 mmol/L,油酸与棕榈酸的比例为2:1)或/和吡格列酮(0.1、1或10μmol/l)干预细胞1小时或48小时。结果显示,FFAs干预一小时,阴性对照组的GSIS显著增加,但转染GPR40shRNA的细胞GSIS未见明显增加。FFAs干预48小时后,GPR40shRNA细胞与阴性对照组GSIS均显著下降。吡格列酮与FFAs共同干预48小时,阴性对照组GSIS较FFAs干预48小时显著增加,但GPR40shRNA组未见显著变化。本研究第三部分探讨FFAs是否通过GPR40影响βTC6细胞PDX-1和GLUT2的表达,吡格列酮对脂毒性的保护作用是否通过GPR40的介导而影响βTC6细胞PDX-1及GLUT2的表达。分别以0.5 mmol/L FFAs干预1小时或48小时,或0.5 mmol/L FFAs与10μmol/l吡格列酮共同干预细胞48小时。结果显示,FFAs干预1小时显著增加阴性对照组PDX-1和GLUT2的表达,而转染GPR40shRNA的细胞PDX-1和GLUT2的表达未见明显增加。FFAs干预48小时后,转染GPR40shRNA的细胞与阴性对照组PDX-1和GLUT2的表达均下降。吡格列酮与FFAs共同干预48小时,阴性对照组PDX-1和GLUT2的表达较FFAs干预48小时显著增加,但GPR40shRNA组未见显著变化。综合上述结果,GPR40介导了FFAs短期作用下β细胞GSIS的增加以及PDX-1和GLUT2表达的上调,但未介导FFAs的慢性脂毒性作用。而吡格列酮对β细胞脂毒性的改善则可能部分通过GPR40的作用增加PDX-1和GLUT2的表达,从而改善GSIS。更多还原

【Abstract】 Free fatty acids (FFAs) acutely stimulate insulin secretion from pancreaticβ-cells, whereas impairβ-cell function following long term exposure. The FFAs receptor, G-protein-coupled receptor 40 (GPR40) has been proposed to mediate insulin secretion, hence to play an important pathophysiological role in type 2 diabetes mellitus. Despite intensive research efforts, the physiological role of GPR40 still remains unclear. In order to gain a better understanding for GPR40, this study was conducted to investigate the role of GPR40 in pancreatic islet cells, including the effect of GPR40 on both short and long-term FFAs on GSIS, the relationship between GPR40 and FFAs mediated expression of PDX-1 and GLUT2 as well as the GPR40 contribution to the thiazolidinedione reverse role on lipotoxicity ofβ-cells.The first part of this study was to establishβTC6 cell line stably expressing GPR40shRNA. Therefore two complementary 55-mer siRNA template oligonucleotides encoding GPR40 short hairpin RNAs (shRNAs) were designed and ligated with linearized pSilencer 4.1-CMV neo siRNA expression vector. Then the sequence was further identified by sequencing from both sides. The plasmid was transfected intoβTC6 cells using lipofectamine method. The clonedβTC6 cells were selected by G418. RT-PCR and western blot were used to detecte the expression of GPR40. The results showed that the recombinant plasmid knocked down GPR40 mRNA and protein inβTC6 cells.βTC6 cell line stably expressing GPR40shRNA were established.The second part of this study was to determine the contribution of GPR40 to short- or long-term effects of FFAs and pioglitazone on glucose stimulated insulin secretion (GSIS) inβTC6 cells. Cells were then incubated in FFAs (0.25,0.5,or 1 mmol/L, a 2:1 mixture of oleate: palmitate) or/and pioglitazone(0.1, 1, or 10μmol/l) for 1 h or 48 h. Results showed that 1-h exposure to FFAs significantly enhanced GSIS in pSilencer-control transfected cells, but not in the cells transfected with GPR40shRNA. While 48-h exposure to FFAs significantly impaired GSIS in pSilencer-control transfected cells as well as the cells transfected with GPR40shRNA. Furthermore, pioglitazone enhanced insulin secretion in pSilencer-control transfected cells exposed to FFAs for 48 h, but not in the cells transfected with GPR40shRNA.The third part of this study was to determine the contribution of GPR40 to short- or long-term effects of FFAs and pioglitazone on the expression of PDX-1 and GLUT2 inβTC6 cells. Cells were incubated in 0.5 mmol/L FFAs for 1 h or 48 h, or 10μmol/l pioglitazone or combination of 0.5 mmol/L FFAs and 10μmol/l pioglitazone for 48 h. Results showed that 1-h exposure to FFAs significantly increased expression of PDX-1 and GLUT2 in pSilencer-control transfected cells, but not in the cells transfected with GPR40shRNA. While 48-h exposure to FFAs significantly decreased expression of PDX-1 and GLUT2 in pSilencer-control transfected cells as well as the cells transfected with GPR40shRNA. Furthermore, pioglitazone increased expression of PDX-1 and GLUT2 in pSilencer-control transfected cells exposed to FFAs for 48 h, but not in the cells transfected with GPR40shRNA.These results indicate that GPR40 mediates the short-term effects of FFAs on GSIS and the expression of PDX-1 and GLUT2, but does not mediate the chronic lipotoxicity onβ-cells. The reverse role of pioglitazone on lipotoxicity ofβ-cells may be related to GPR40.更多还原