【作者】 刘丹；

【导师】 黄起壬；

【作者基本信息】 南昌大学 ， 药学， 2017， 硕士

【摘要】 目的:本课题拟构建SUMO特异性蛋白酶1(SENP1)过表达腺病毒,感染至人脐静脉内皮细胞(HUVECs),探究高表达的SENP1能否介导PPARγ1K77位点发生去SUMO-1化,SENP1介导的PPARγ1K77去SUMO-1化对高糖高脂应激诱导HUVECs胰岛素抵抗(IR)的影响及其机制。方法:(1)构建SENP1过表达腺病毒(Ad-SENP1),筛选最佳感染条件。选用2E+8PFU、2E+7PFU、2E+6PFU三个不同滴度的阴性对照腺病毒(Vehicle)感染内皮细胞48h,根据细胞感染效率及状态筛选出最佳感染滴度;参照阴性对照腺病毒感染的最佳滴度,将Ad-SENP1感染至细胞,使病毒数与细胞数的比值(即MOI)分别为2.5,5,10,20。48h后检测Ad-SENP1的表达水平,筛选出最佳感染条件。(2)PPARγ1K77去SUMO-1化对高糖高脂诱导HUVECs胰岛素抵抗的影响。实验分组:(1)正常组(Ctrl);(2)胰岛素抵抗组(IR);(3)IR+阴性对照组(Vehicle);(4)IR+SENP1过表达腺病毒组(Ad-SENP1)。待细胞长至约60%融合度时,Vehicle组和Ad-SENP1组分别感染相应腺病毒。除Ctrl组外,所有组均换成含葡萄糖(HG,22 mmol/L)和棕榈酸(PA,0.25mmol/L)的DMEM培养基,诱导HUVECs 24h为胰岛素抵抗模型。24h后,每组均换成含胰岛素(Insulin)100nM的DMEM培养基,孵箱内孵育30min。Western blotting法检测SENP1、免疫共沉淀法(Co-IP)检测PPARγ1SUMO-1化的水平;收集细胞上清液检测NO和AngII的水平。(3)PPARγ1K77去SUMO-1化改善高糖高脂诱导血管内皮细胞胰岛素抵抗的机制:荧光倒置显微镜下观察细胞内活性氧(ROS)的水平;细胞内线粒体膜电位的改变;Western blotting法检测IKK、IKK-pS176、免疫共沉淀法(Co-IP)检测IKK与PIAS1的相互作用;Western blotting法检测胰岛素信号通路PI3K/AKT/eNOS的表达水平。结果:(1)2E+7PFU滴度的阴性对照腺病毒感染细胞48h后,细胞状态最好,感染效率最高;Western blotting检测结果显示,细胞内SENP1过表达,腺病毒构建成功;随着MOI值的递增,SENP1过表达越来越明显,结合显微镜下观察到的细胞状态,确定MOI为10时感染效果最佳。(2)PPARγ1K77去SUMO-1化能改善高糖高脂应激下HUVECs胰岛素抵抗的状态。感染腺病毒并高糖高脂诱导HUVECs致IR,结果显示,与Vehicle组比较,Ad-SENP1组PPARγ1SUMO-1化水平显著下调、NO水平显著上升、AngⅡ水平显著下降,表明SENP1过表达可使PPARγ1K77位点实现去SUMO-1化,并且PPARγ1K77去SUMO-1化可改善高糖高脂诱导的血管内皮细胞IR。(3)PPARγ1K77去SUMO-1化改善高糖高脂诱导血管内皮细胞胰岛素抵抗的作用机制。结果显示,与Vehicle组比较,Ad-SENP1组ROS生成量明显减少,线粒体膜电位崩塌明显减轻;Western blotting结果显示Ad-SENP1组IKK-pS176水平显著下调;免疫共沉淀结果显示,IKK与PIAS1的相互作用也随之减弱;Western blotting结果显示,PPARγ1K77去SUMO-1化可解除高糖高脂应激引起的PI3K/AKT/eNOS信号通路的抑制状态。结论:SENP1过表达可使PPARγ1K77位点实现去SUMO-1化;PPARγ1K77去SUMO-1化可改善高糖高脂应激诱导的HUVECs胰岛素抵抗,其机制可能与ROS生成减少、IKK-pS176活性降低、IKK与PIAS1的相互作用减弱以及PI3K/Akt/eNOS信号通路抑制状态得到解除有关。更多还原

【Abstract】 Objective: In this study,we sought to investigate the effect of SENP1-mediated PPARγ1 deSUMOylation on insulin resistance induced by high glucose and palmitic acid in endothelial cells and finally probe its underlying mechanisms.Methods:(1)HUVECs were transfected with vehicle vectors in various concentrations(2E+8PFU,2E+7PFU,2E+6PFU)for 48 h to select an optimal titer.Then,Ad-SENP1 vectors were introduced into endothelial cells with different multiplicities of infection(2.5,5,10,20)for 48 h.The expression levels of SENP1 were measured by western blotting.(2)In this section,four groups were divided,namely Control group,IR group,Vehicle group and Ad-SENP1 group.Briefly,when HUVECs were grown to 60% confluence,Vehicle group and Ad-SENP1 group were infected with the corresponding adenovirus vectors respectively.Then,all groups except Control were replaced with glucose and PA medium(22mmol/L glucose,0.25mmol/L palmitic acid)for 24 h in order to establish the insulin resistance(IR)model.After that,all groups were substituted with fresh L-DMEM containing insulin(100nmol/L)for 30 minutes.Immunocoprecipitation(Co-IP)and western blotting assays were used to detect the SUMO-1 levels of PPARγ1.The levels of NO and AngII were measured to validate the improvement of SENP1-mediated PPARγ1deSUMOylation on insulin resistance induced by high glucose and palmitic acid in endothelial cell.(3)To probe its underlying mechanisms,fluorescence microscope and flow flurocytometry were utilized to test the levels of reactive oxygen species(ROS)and mitochondrial membrane potential.Besides,IKK and IKK-pS176 were detected by western blotting analysis.Co-IP and western blotting assays were used to detect the interactions between IKK and PIAS1.Finally,target proteins of PPARγ1including PI3 K,AKT,AKT-pS473 and eNOS were detected by western blotting analysis.Results:(1)Higher infection efficiency and better cell shape were shown in HUVECs transfected with vehicle vectors at 2E+7PFU concentration than those at other concentrations.The over-expression of SENP1 by western blotting analysis demonstrated that Ad-SENP1 vectors was constructed successfully.When themultiplicity of infection was 10,the effect was optimal.(2)SENP1-mediated PPARγ1 deSUMOylation can improve the insulin resistance induced by high glucose and palmitic acid in endothelial cells.After the infection of adenovirus vectors and establishment of the insulin resistance(IR)model,the results were as follows.Compared with Vehicle group,the levels of SUMOylated PPARγ1 were reduced in Ad-SENP1 group.Besides,the level of NO increased significantly,yet the level of AngII decreased remarkably in Ad-SENP1 group.Together,overexpression of SENP1 can mediate PPARγ1 deSUMOylation and improve the insulin resistance induced by high glucose and palmitic acid in endothelial cells.(3)Compared with Vehicle group,the ROS production was significantly reduced and the mitochondrial membrane potential showed attenuated collapse in Ad-SENP1 group.In addition,the interaction between IKK and PIAS1 was weakened as well.Moreover,the results of western blotting revealed that the expression of IKK-pS176 decreased notably while the levels of PI3 K,AKT-pS473 and eNOS were significantly increased.Conclusion: Overexpression of SENP1 can mediate PPARγ1 deSUMOylation and improve the insulin resistance induced by high glucose and palmitic acid in endothelial cells.This may be due to reducing the levels of ROS,inhibiting the activity of IKK and interaction between IKK and PIAS1 and resultantly activating the PI3K/Akt/eNOS pathway.更多还原