【作者】 刘腾飞；

【导师】 邓平；

【作者基本信息】 南华大学 ， 内科学， 2014， 硕士

【摘要】 目的：通过培养小鼠3T3-L1细胞,诱导分化为成熟脂肪细胞,用高糖高胰岛素诱导建立脂肪细胞胰岛素抵抗模型,观察不同浓度可溶性环氧化物水解酶抑制剂t-AUCB对胰岛素抵抗脂肪细胞的葡萄糖摄取率、TNF-α、和脂联素分泌、及GLUT-4mRNA表达和蛋白合成的影响。方法：1、小鼠3T3-L1细胞的诱导和分化；2、脂肪细胞胰岛素抵抗模型的建立：采用高糖高胰岛素培养基诱导培养脂肪细胞,并通过检测培养后脂肪细胞对葡萄糖的摄取率鉴定模型是否成功；3、将建立模型后的脂肪细胞共分为5组(每组含脂肪细胞约2×104个细胞),其中1组为对照组,其余4组为干预组,分别采用不同浓度(0,1,10,50,100μmol/L) t-AUCB干预培养24h,同时,取普通培养的脂肪细胞作为空白对照组。分别提取上清液,收集脂肪细胞；4、采用葡萄糖氧化酶-过氧化物酶法测定各组上清液中葡萄糖的浓度,计算葡萄糖摄取率；5、采用ELISA法检测各组上清液脂联素、TNF-α水平；6、采用Real time-PCR法检测各组脂肪细胞GLUT4mRNA表达,采用免疫印迹法检测各组脂肪细胞GLUT4蛋白水平。结果：1.3T3-L1前脂肪细胞诱导分化为成熟脂肪细胞,经油红0染色后,光镜下可见分化成熟的脂肪细胞内的脂滴被染成橘红色。2.高糖高胰岛素培养液诱导后的脂肪细胞葡萄糖摄取率与普通培养液培养的脂肪细胞葡萄糖摄取率相比有显著差异(3±1.9%VS40±1.2%,P<0.05)。3.采用1,10,50,100μmol/L的t-AUCB干预后,脂肪细胞对葡萄糖摄取率分别为7±1.5%、17±1.2%、32±1.6%、36±0.9%,均明显高于对照组(3±1.4%),差异具有显著性意义(均P<0.05),且呈浓度依赖性增高(均P<0.05)。空白对照组葡萄糖摄取率显著高于对照组(42±1.7%VS3±1.4%,P<0.05)。4.采用1,10,50,100μmol/L的t-AUCB干预后,脂肪细胞上清液脂联素浓度水平分别为,0.64±0.07.1.30±0.11、1.66±O.05、2.33±0.04ng/mL,均明显高于对照组(0.33±0.02ng/mL),差异具有显著性意义(均P<0.05),且呈浓度依赖性增高(均P<0.05)。空白对照组脂联素浓度水平显著高于对照组(3.04±0.07VS0.33±0.02ng/mL,P<0.05)。5.采用1,10,50,100μmol/L的t-AUCB干预后,脂肪细胞上清液TNF-a浓度水平分别为,163.30±3.24、125.52±2.08、93.93±2.62、49.65±1.20pg/mL,均明显低于对照组(182.34±4.89pg/mL),差异具有显著性意义(均P<0.05),且呈浓度依赖性降低(均P<0.05)。空白对照组TNF-a浓度水平显著低于对照组(8.06±1.80VS182.34±4.89pg/mL,P<0.05)6.采用1,10,50,100μmol/L的t-AUCB干预后,脂肪细胞GLUT-4mRNA表达RQ值分别为1.28±0.41、2.00±0.08、2.42±0.12、3.41±0.21,均明显高于对照组(1.12±0.18),差异具有显著性意义(均P<0.05),且呈浓度依赖性增高(均P<0.05)。空白对照组GLUT-4mRNA表达RQ值显著高于对照组(3.67±0.26VS1.12±0.18,P<0.05)。7.采用1,10,50,100μmol/L的t-AUCB干预后,脂肪细胞GLUT-4蛋白的光密度值(GLUT-4/GAPDH)分别为0.329±0.118、0.472±0.123、0.623±0.172、0.756±0.112,均明显高于对照组(0.312±0.115),差异具有显著性意义(均P<0.05),且呈浓度依赖性增高(均P<0.05)。空白对照组脂肪细胞GLUT-4蛋白的光密度值(GLUT-4/GAPDH)显著高于对照组(0.810±0.121VS0.312±0.115,P<0.05)。结论：1.可溶性环氧化物水解酶抑制剂t-AUCB可促进胰岛素抵抗脂肪细胞GLUT-4蛋白表达,从而增加葡萄糖摄取,改善脂肪细胞胰岛素抵抗。2.可溶性环氧化物水解酶抑制剂t-AUCB可呈浓度依耐性促进胰岛素抵抗脂肪细胞脂联素分泌水平,而降低TNF-a分泌水平,改善胰岛素抵抗脂肪细胞分泌功能。更多还原

【Abstract】 Mouse3T3-L1Preadipocytes were induced to differentiate into adipocytes.Then, adipocytes Insulin resistance(IR) model were created by high glucose and high High method.Our objective was to investigate the the effect of t-AUCB(trans--[4-(3-adamantan-1-y1-ureido)-cyclohexyl]-benzoic acid), a selective soluble epoxide hydrolase inhibitors, on glucose uptake and tumor necrosis factor-a (TNF-α) and adiponectin secretion and glucose transporter-4(GLUT-4) mRNA, protein expression in Insulin resistance adipocytes.Methods1.Mouse3T3-L1Preadipocytes were induced to differentiate into adipocytes;2. adipocyte insulin resistance model building:adipocytes were cultured with high glucose and high insulin method, and the model was identified through detection glucose uptake of adipocytes cultured;3. The adipocytes modeled after divided into5groups (including adipocytes for about2×104Cells), of which one was the control group, and the remaining four groups were the intervention groups, respectively, using different concentrations (0,1,10,50,100μmol/L)) t-AUCB intervention cultured24h, at the same time, take the common culture of fat cells as the control group. Supernatants were extracted, collected adipocytes;4. The concentration of glucose in the supernatant of each group was determined by glucose oxidase-peroxidase method;5.The levels of adiponectin and TNF-α in the supernatant were Measured by ELISA.6.GLUT4protein were Measured by Western Blot and GLUT4mRNA by Real time-PCR. Results1.3T3-L1preadipocytes were induced to differentiate into mature adipocytes. After oil red O staining, we can found that the fat droplets in mature adipocytes weres dyed orange Under the light microscope.2. There are significant differences between the glucose uptake rate of adipocytes cultured by high glucose and high insulin and cultured by general culture medium.(3±1.9%VS40±1.2%, P<0.05).3. After using1,10,50,100μmol/L of t-AUCB intervention, the glucose uptake rate of adipocyte respectively was7±1.5%,17±1.2%,32±1.6%,36±0.9%, significantly higher than the control group (3±1.4%), the difference was significant (all P<0.05), and showed a dose-dependent manner (all P<0.05). Blank control group compared with control group also significantly different (42±1.7%VS3±1.4%, P<0.05).4. After using1,10,50,100μmol/L of t-AUCB intervention,the levels of the supernatants adiponectin Concentration of adipocyte respectively was,0.64±0.07,1.30±0.11,1.66±0.05,2.33±0.04ng/mL, were significantly higher than control group (0.33±0.02), the difference was significant (all P<0.05), and showed a dose-dependent manner (all P<0.05). Blank control group compared with control group also significantly different (3.04±0.07VS0.33±0.02, P <0.05).5. After using1,10,50,100μmol/L of t-AUCB intervention, the levels of the supernatants TNF-a Concentration of adipocyte respectively were163.30±3.24,125.52±2.08,93.93±2.62,49.65±1.20pg/mL, were significantly lower than the control group (182.34±4.89pg/mL), the difference was significant (all P<0.05), and showed a dose-dependent manner (all P<0.05). Blank control group compared with control group also significantly different (8.06±1.80VS182.34±4.89pg/mL, P<0.05)6. After using1,10,50,100μmol/t-AUCB intervention, the RQ values of GLUT-4mRNA expression of adipocyte respectively was1.28±0.41,2.00±0.08,2.42±0.12,3.41±0.21, significantly higher than the control group (1.12± 0.18), the difference was significant (all P<0.05), and showed a dose-dependent manner (all P<0.05). Blank control group compared with control group also significantly different (3.67±0.26VS1.12±0.18, P<0.05).7. After using1,10,50,100μmol/L of t-AUCB intervention, the GLUT-4protein optical density (GLUT-4/GAPDH) of adipocyte respectively were0.329±0.118,0.472±0.123,0.623±0.172,0.756±0.112, significantly higher than the control group (0.312±0.115), the difference was significant (all P<0.05), and dose-dependent manner (all P<0.05). Blank control group compared with control group also significantly different (0.810±0.121VS0.312±0.115, P <0.05).Conclusions1. Epoxide hydrolase inhibitors t-AUCB may promote the GLUT-4protein expression, and glucose uptake in Insulin resistance Adipocytes By a way of dose-dependent manner, which improved the insulin resistance in Insulin resistance adipocytes.2. Epoxide hydrolase inhibitors t-AUCB may increase the secretion of adiponectin while reducing the secretion of TNF-a levels, By a way of dose-dependent manner in Insulin resistance Adipocytes,which improved Insulin resistance adipocytes secretory function.更多还原