【作者】 张敏；

【导师】 朱大龙；

【作者基本信息】 南京大学 ， 临床医学（专业学位）， 2015， 硕士

【摘要】 第一部分SGK1抑制剂改善糖代谢紊乱的作用研究目的:在前期研究的基础上,进一步验证SGK1抑制剂对高脂诱导肥胖模型小鼠糖代谢紊乱的改善作用,初步探讨该作用是否与改善脂肪组织功能紊乱有关。方法:1、C57/BL6雄性小鼠,高脂喂养12周后,挑选肥胖及糖尿病的成模小鼠。按空腹体重和空腹血糖随机分为:高脂组、SGK1抑制剂组,同时设同品种同周龄大小的雄性小鼠并喂以普通饲料(普食组),干预12周。实验过程中,每2周观察空腹体重、空腹血糖,干预第12周后行口服葡萄糖耐量试验(OGTT)、腹腔葡萄糖耐量试验(IPGTT)及腹腔胰岛素耐量试验(IPITT)。实验结束时,留取各组小鼠的血清及脂肪组织。2、取课题组前期动物实验中留取的db/m野生组、db/db对照组及SGK1抑制剂组小鼠的脂肪组织,用real time-PCR的方法检测各组小鼠脂肪组织中脂肪细胞因子adiponectin、MCP-1、PAI-1、IL-6的mRNA表达水平。3、取课题组前期动物实验中留取的db/m野生组、db/db对照组及SGK1抑制剂组小鼠的血清,用ELISA方法检测各组小鼠血清中adiponectin及MCP-1水平。结果:1、SGK1抑制剂干预治疗后糖代谢水平的评估:(1)空腹血糖(FBG):与高脂组相比,SGK1抑制剂组空腹血糖第2周起即下降,且6、10、12周时有统计学差异(p<0.05);(2)口服葡萄糖耐量试验(OGTT):与高脂组相比,SGK1抑制剂组30min血糖下降有统计学差异(p<0.05);(3)腹腔葡萄糖耐量试验(IPGTT):与高脂组相比,SGK1抑制剂组Omin、120min血糖下降有统计学差异(p<0.05);(4)腹腔胰岛素耐量试验(IPITT):与高脂组相比,SGK1抑制剂组60min、120min血糖下降有统计学差异(p<0.05)。2、SGK1抑制剂干预治疗后,脂肪组织中相关细胞因子mRNA表达水平变化:与db/db对照组相比,SGK1抑制剂组小鼠脂肪组织中adiponectinmRNA表达没有明显变化,但MCP-1、PAI-1、IL-6的mRNA表达均有下降趋势。3、SGK1抑制剂干预治疗后,血清中相关细胞因子水平变化:与db/db对照组相比,SGK1抑制剂组小鼠血清中adiponectin的水平有升高趋势,MCP-1的水平有下降趋势。结论:SGK1抑制剂干预治疗可一定程度改善肥胖胰岛素抵抗模型小鼠糖代谢紊乱,该作用可能部分与改善脂肪组织功能紊乱有关。第二部分SGK1表达及活性增加可能介导3T3-L1脂肪细胞胰岛素抵抗目的:明确在3T3-L1脂肪细胞中,SGK1表达及活性增加是否诱导脂肪细胞胰岛素抵抗,SGK1抑制剂是否可抑制该作用,并进一步研究SGK1与NF-κB信号通路的相关性。方法:1、3T3-L1前脂肪细胞,诱导分化成熟后,地塞米松不同浓度、不同时间干预,干预结束后通过Western blot方法检测各组脂肪细胞中SGK1、p-SGK1、IRS-1、IRS-2及NF-κB信号通路相关蛋白表达水平;地塞米松+SGK1抑制剂共同干预3T3-L1脂肪细胞,通过Western blot方法检测各组细胞中SGK1、p-SGK1、IRS-1、IRS-2及NF-κB信号通路相关蛋白表达水平。2、组成性激活突变S422DSGK1过表达慢病毒转染3T3-L1前脂肪细胞,诱导分化成熟后,加或不加胰岛素刺激,通过Western blot方法检测各组脂肪细胞中SGK1、AKT及P-AKT的蛋白表达水平。结果:1、与对照组相比,地塞米松浓度及时间依赖的促进3T3-L1脂肪细胞中SGK1及p-SGK1的蛋白表达,下调IRS-1、IRS-2的蛋白表达;地塞米松+SGK1抑制剂共同干预后,与地塞米松组相比,地塞米松+SGK1抑制剂组IRS-1、IRS-2的下调作用被部分逆转。2、慢病毒转染成功、胰岛素刺激后,与阴性病毒对照组相比,SGK1过表达慢病毒转染组P-AKT的蛋白水平明显下降。3、与对照组相比,地塞米松浓度及时间依赖的促进3T3-L1脂肪细胞中IκBα磷酸化并降低IκBα蛋白表达;地塞米松+SGK1抑制剂共同干预后,与地塞米松组相比,地塞米松+SGK1抑制剂组IκBα的下调作用被部分逆转。结论:在3T3-L1脂肪细胞中,SGK1表达及活性增加可能介导脂肪细胞胰岛素抵抗,SGK1抑制剂可部分抑制该作用;SGK1表达及活性增加同时激活NF-κB信号通路,该作用是否与介导脂肪细胞胰岛素抵抗相关需进一步研究证实。更多还原

【Abstract】 Patr one Studies on the treatment effect of SGK1 inhibitors on improving glucose metabolism disordersObjective:On the basis of the previous studies,and further to validate the treatment effect of SGK1 inhibitors on improving glucose metabolism disorders in the model mice of the high-fat induced obesity,and to explore whether the possible mechanism is due to the effect of improving the adipose tissue dysfunction.Methods:1、C57/BL6 male mice,after feeding high-fat food for 12 weeks,choose the model mices of obesity and diabetes.According to the fasting weight and the fasting blood glucose,the model mices were randomly divided into:high-fat group,SGK1 inhibitor group,at the same time set up the male mices with the same varietie and the same weeks of age,feeding with the ordinary fodder(named normal diet group).The intervention lasted for 12 weeks.During the intervention period,fasting blood glucose and fasting body weight were checked every two weeks.After the intervention,oral glucose tolerance test(OGTT)、intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)were conducted.At the end of the experiment,the mice were killed.Blood serum and adipose tissues were collected and stored for further examation.2、The mRNA expression levels of the adipokines,such as adiponectin、MCP-1、PAI-1 and IL-6 were detected in the adipose tissues of the mices using quantitative real-time PCR,these adipose tissues were collected from the mices of db/m wild group、db/db control group and SGK1 inhibitor group during our previous animal experiments。3、The expression levels of the adiponectin and MCP-1 were detected in the blood serum of the mices using ELISA kit,the blood serum were collected from the mices of db/m wild group、db/db control group and SGKl inhibitor group during our previous animal experiments。Results:The carbohydrate metabolism evaluation after the treatment of SGK1 inhibitors:(1)Fasting blood glucose:Compared to high-fat group,SGK1 inhibitor group had lower blood glucose level since the second week,and a significant difference at the 6、10 and 12 week(p<0.05);(2)OGTT:Compared to high-fat group,SGK1 inhibitor group had lower blood glucose level at 30min(p<0.05);(3)IPGTT:Compared to high-fat group,SGK1 inhibitor group had lower blood glucose level at 0、120min(p<0.05);(4)IPITT:Compared to high-fat group,SGK1 inhibitor group had lower blood glucose level at 60、120min(p<0.05).2、After the treatment of SGK1 inhibitor,the mRNA expression levels of adipose tissue adipokines:Compared to the db/db control group,the mRNA expression level of adipose tissue adiponectin did not change significantly in the SGK1 inhibitor group,but the mRNA expression level of MCP-1、PAI-1 and IL-6 were significantly decreased.3、After the treatment of SGK1 inhibitor,the expression levels of blood serum cytokines:Compared with the db/db control group,the serum adiponectin expression tended to increase and the expression of MCP-1 tended to decrease in SGK1 inhibitor group.Conclusion:SGK1 inhibitors treatment can significantly improve hyperglycemia in obese mice with insulin resistance,the possible mechanisms may be partially due to its effect of improving adipose dysfunction.Patr two The increased expression and activity of SGK1 may mediate the insulin resistance in 3T3-L1 adipocytesObjective:To definite whether the increased expression and activity of SGK1 will induce the insulin resistance in 3T3-L1 adipocyte,whether SGK1 inhibitors could block this effect,and to further study the correlation of SGKI and NF-κB signaling pathway.Methods:1、3T3-L1 preadipocytes,induced differentiation and maturation,and then incubated with dexamethasone in different concentration and different time.After the intervention,the protein expression levels of SGKl、p-SGK1、IRS-1、IRS-2 and the related protein of NF-kB signaling pathway were measured by western blot.2、Constitutively active mutant S422DSGK1 overexpression lentivirus were transfected into 3T3-L1 preadipocytes,induced differentiation and maturation,with or without insulin stimulation,and then detect the protein expression levels of SGKI、AKT and P-AKT in adipocytes using Western Blot.Results:1、Compared with the control group,Dexamethasone increased the protein expression of SGK1、P-SGK1,and downergulated the protein expression of IRS-1、IRS-2 in a concentration and time dependent manner in 3T3-L2 adipocytes.Compared with the dexamethasone group,the downregulation of IRS-1 and IRS-2 protein expression is partially reversed in dexamethasone plus SGK1 inhibitor intervention group.2、After the Lentivirus transfection successfully and with insulin stimulation,the P-AKT protein levels were decreased significantly in SGK1 overexpression lentivirus group compared with the control group.3、Compared with the control group,Dexamethasone increased the IκBα phosphorylation and reduced the IκBα protein expression in a concentration and time dependent manner in 3T3-L1 adipocytes.Compared with the dexamethasone group,the downregulation of IκBαprotein expression is partially reversed in dexamethasone plus SGK1 inhibitor intervention group.Conclusion:The increased expression and activity of SGK1 may mediate the insulin resistance in 3T3-L1 adipocytes,SGK1 inhibitors can block this effect partially.The increased expression and activity of SGK1 activate the NF-κB signaling pathway,Whether this effect is associated with adipocyte insulin resistance need to be further studied.更多还原