环氧二十碳三烯酸对内皮细胞内质网应激的作用及机制

发布时间：2018-01-18 09:21

本文关键词：环氧二十碳三烯酸对内皮细胞内质网应激的作用及机制　出处：《华中科技大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:探讨花生四烯酸细胞色素P450表氧化酶代谢产物环氧二十碳三烯酸(Epoxyeicosatrienoic acids,EETs)对内皮细胞内质网应激(Endoplasmic reticulum stress,ERS)的作用及机制。方法:1、用10μM衣霉素(TM)诱导2h-11细胞和脐静脉内皮细胞(HUVECs)产生内质网应激,应用Western Blot检测细胞内质网应激相关蛋白p-PERK、Grp78、Xbp-1等水平的表达。 2、分别应用50nM,250nM和1μM的三种EETs(8,9-EET、11,12-EET和14,15-EET)处理内皮细胞,应用Western Blot检测内质网应激相关蛋白p-PERK、Grp78、Xbp-1等的表达和内质网促凋亡蛋白CHOP、p-c-Jun和有活性的caspase 12的表达;应用流式细胞仪检测内皮细胞的凋亡情况;应用CCK-8检测内皮细胞的活性。 3、用相关信号通路抑制剂(G蛋白、Gs蛋白、腺苷酸环化酶、蛋白激酶A、蛋白激酶G、PI3K、EGFR、MEK和PPAR-γ抑制剂)处理2h-11细胞和HUVECs,应用Western Blot检测内质网应激相关蛋白Grp78的表达。结果:1、Western Blot结果显示,2h-11细胞在TM处理4h时,内质网应激相关蛋白p-PERK、Grp78、Xbp-1等水平明显增加。脐静脉内皮细胞在TM处理8h时,p-PERK、Grp78、Xbp-1等水平明显增加;有显著统计学意义(P0.05)。 2、8,9-EET、11,12-EET和14,15-EET可以明显减少TM诱导的2h-11细胞Grp78水平,且11,12-EET较其它两种EETs,能明显的减少p-PERK的水平。11,12-EET对Xbp-1和p-PERK的表达具有浓度依赖性。而14,15-EET与8,9-EET、11,12-EET和TM+DMSO相比,均能降低Grp78的表达水平,且14,15-EET对Xbp-1的表达具有浓度依赖性。流式细胞术和CCK-8检测结果显示:在2h-11细胞中,TM+DMSO组和TM+11,12-EET组的凋亡百分数分别为:(35.210±2.887)%和(15.690±1.732)%,细胞的凋亡百分数并增加细胞活性(P0.05);在HUVECs中,TM+DMSO组和TM+14,15-EET组的凋亡百分数分别为:(41.673±3.756)%和(22.970±1.732)%,细胞活性分别为1.131±0.028和1.594±0.094,这表明,14,15-EET能明显降低HUVECs的凋亡百分数并增加细胞活性(P0.05)。 3、G蛋白抑制剂GDP-β-s、Gs蛋白抑制剂NF-449、腺苷酸环化酶(AC)抑制剂SQ22536、蛋白激酶A(PKA)抑制剂H-89和蛋白激酶G(PKG)抑制剂KT-5823与TM+EETs组相比,Grp78水平明显增加(P0.05)。而EGFR、MEK、PI3K和PPAR-γ抑制剂与TM+EETs组相比,Grp78水平无明显改变,无统计学差异(P0.05)。结论:1、外源性的EETs可以改善由衣霉素诱导的内皮细胞的内质网应激和细胞凋亡,并增加细胞的活性。 2、外源性的EETs改善由衣霉素诱导的内皮细胞的内质网应激的机制可能是通过G蛋白偶联受体,进而激活PKA和PKG。 3、外源性的EETs改善由衣霉素诱导的内皮细胞的内质网应激的机制可能不通过激活PI3K、EGFR、MEK和PPAR-γ。
[Abstract]:Objective: to investigate the metabolite of arachidonic acid cytochrome P450 epioxidase epoxide 20 carbotrienoic acid (Epoxyeicos atrienoic acids). The effect and mechanism of eETs on endoplasmic reticulum stress (Endoplasmic reticulum stress) in endothelial cells. Methods the endoplasmic reticulum (ER) stress was induced in 2h-11 cells and HUVECs by 10 渭 M TMM. The expression of cytoplasmic reticulum stress-related protein (p-PERKN) Grp78 (Xbp-1) was detected by Western Blot. 2. The endothelial cells were treated with 50nM 250nM and 1 渭 M EETs89-EET-1112-EET and 1415-EETs, respectively. Western Blot was used to detect the expression of the ER stress-related protein p-PERKN Grp78 Xbp-1 and the endoplasmic reticulum apoptotic protein (CHOP). The expression of p-c-Jun and active caspase 12; Apoptosis of endothelial cells was detected by flow cytometry. The activity of endothelial cells was detected by CCK-8. 3. The related signal pathway inhibitors (G protein G protein, adenylate cyclase, protein kinase A, protein kinase GN PI3K) EGFR were used. 2 h-11 cells and HUVECs were treated with MEK and PPAR- 纬 inhibitor. The expression of ER stress-related protein Grp78 was detected by Western Blot. Results the results of Western Blot showed that the endoplasmic reticulum stress-related protein p-PERKN Grp78 was treated with TM for 4 h. The level of Xbp-1 was significantly increased, and the level of p-PERKN Grp78 Xbp-1 was significantly increased at 8 h after TM treatment. There was significant statistical significance (P 0.05). The levels of Grp78 in 2h-11 cells induced by TM were significantly decreased by TM-induced Grp78 levels in 2h-11 cells. Compared with the other two EETs, 12-EET significantly reduced the level of p-PERK. 1112-EET was concentration-dependent on the expression of Xbp-1 and p-PERK. Compared with 89-EET-1112-EET and TM DMSO, 15-EET can decrease the expression of Grp78, and 14. The results of flow cytometry and CCK-8 analysis showed that in 2h-11 cells, TM DMSO and TM 11 were detected. The percentage of apoptosis in 12-EET group was 35.210 卤2.887% and 15.690 卤1.732%, respectively. The percentage of apoptosis and the activity of P0.05G were increased. TM DMSO group and TM 14 in HUVECs. The percentages of apoptosis in 15-EET group were 41.673 卤3.756% and 22.970 卤1.732%, respectively. The cell activity was 1.131 卤0.028 and 1.594 卤0.094, respectively, which indicated that the activity of Con 14 was 1. 131 卤0. 028. 15-EET could significantly decrease the percentage of apoptosis of HUVECs and increase the activity of P0. 05%. 3, GDP- 尾 -songgs protein inhibitor NF-449, adenylate cyclase acid) inhibitor SQ22536. Protein kinase (PKA) inhibitor H-89 and protein kinase Gluten PKG inhibitor (KT-5823) were compared with TM EETs group. The level of Grp78 increased significantly (P 0.05), but the levels of PI3K and PPAR- 纬 inhibitor of EGFR EETs did not change significantly compared with those of TM EETs group. There was no statistical difference (P 0.05). Conclusion EETs can improve endoplasmic reticulum stress and apoptosis of endothelial cells induced by itamycin, and increase cell activity. 2. The mechanism of exogenous EETs in improving endoplasmic reticulum stress in endothelial cells induced by Ichlamycin may be the activation of PKA and PKG by G protein-coupled receptors. 3The mechanism of exogenous EETs in improving endoplasmic reticulum stress in endothelial cells induced by itamycin may not be mediated by activation of PI3KNEGFRMEK and PPAR- 纬.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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