二甲双胍对软脂酸诱导血管内皮细胞损伤的保护作用研究

发布时间：2018-06-17 03:01

本文选题：二甲双胍 + 内皮细胞　；参考：《贵州医科大学》2017年硕士论文

【摘要】：目的:本实验探讨二甲双胍(Metformin,Met)对软脂酸(palmitic acid,PA)诱导损伤的血管内皮细胞是否具有保护作用,并对其可能发生的机制做进一步研究,为糖尿病合并心血管病患者的治疗提供新思路和靶点。方法:(1)将人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)分为5组,依次为空白对照组(常规细胞培养)、PA组(300μmol/L)、低浓度Met(1mmol/L)+PA联合组、中浓度Met(2mmol/L)+PA联合组、高浓度Met(4mmol/L)+PA联合组,在无血清培养液中培养24小时后用CCK-8法测定各组细胞活性。(2)用蛋白质印迹法(Westenblot)测定各组细胞核因子κB(nuclear factorκB p65,NFκB p65)、细胞间黏附因子(Intercellular cell adhesion molecule 1,ICAM1)、核因子κB抑制蛋白(inhibitor of NF-κB alpha,IκBα)、磷酸化核因子κB抑制蛋白(p-IκBα),腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)和磷酸化腺苷酸活化蛋白激酶(p-AMPK)的蛋白表达。(3)用实时荧光定量PCR(q RT-PCR)的方法测定各组细胞单核细胞趋化因子(Chemokine ligand 2,CCL2)和细胞间黏附因子(ICAM1)的mRNA表达。结果:(1)NFκB p65的蛋白表达量:与对照组相比,PA组表达量明显升高(P0.05);与PA组相比,Met+PA联合组细胞表达量明显降低(P均0.05),其中中浓度Met+PA联合组较其他浓度Met+PA联合组蛋白表达量更低(P均0.05)。(2)p-IκBα的蛋白表达量:与对照组相比,PA组表达量明显升高(P0.05);与PA组相比,Met+PA联合组细胞表达量明显降低(P均0.05),其中中浓度Met+PA联合组较其他浓度Met+PA联合组蛋白表达量更低(P均0.05)。(3)ICAM1的蛋白表达量:与对照组相比,PA组表达量明显升高(P0.05);与PA组相比,Met+PA联合组细胞表达量随Met浓度梯度增高而递减(P均0.05)。(4)p-AMPK的蛋白表达量:与对照组和PA组相比,Met+PA联合组细胞随着Met浓度梯度的增加,其蛋白表达量显著增高(P均0.05),其中中浓度Met+PA联合组较其他浓度Met+PA联合组蛋白表达量更高(P均0.05)。(5)ICAM1的mRNA表达量:与对照组相比,PA组表达量有所增加(P0.05);与PA组相比,Met+PA联合组表达量随Met浓度梯度的升高而减少(P均0.05)。(6)CCL2的mRNA表达量:与对照组相比,PA组表达量显著增加(P0.05);与PA组相比,Met+PA联合组表达量随Met浓度梯度的升高而减少(P均0.05)。结论:二甲双胍可以通过抑制NFκB通路减轻软脂酸诱导的血管内皮细胞炎性反应,降低软脂酸在细胞内引起的细胞粘附因子和趋化因子的表达,对细胞起到一定的保护作用;而在加入二甲双胍的内皮细胞中,腺苷酸活化蛋白激酶被磷酸化激活,它的激活可能与NFκB通路的失活有重要联系。
[Abstract]:Objective: to investigate the protective effect of metformin (Metformin) on the vascular endothelial cells (VEC) induced by palmitic acidinic acid (PAA), and to further study the possible mechanism of metformin (Metformin metformin) on the injury of vascular endothelial cells (VEC) induced by PAA. To provide new ideas and targets for the treatment of diabetic patients with cardiovascular disease. Methods Human umbilical vein endothelial cells (HUVECs) were divided into 5 groups: blank control group (normal cell culture group) (300 渭 mol 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) PA group, medium concentration (2 mmol / L ~ (-1) PA group, and high concentration of Met4 mmol 路L ~ (-1) PA group. After cultured in serum-free medium for 24 hours, the cell activity was measured by CCK-8 method. (2) the nuclear factor 魏 B p65 魏 B p65, intercellular cell adhesion molecule 1 / ICAM1, inhibitor of NF- 魏 B protein inhibitor of NF- 魏 B were detected by Western blot. Protein expression of alpha I 魏 B 伪, phosphorylated nuclear factor- 魏 B inhibitor protein p-I 魏 B 伪, adenosine monophosphate activated protein kinase (AMPK) and phosphorylated adenosine activated protein kinase p-AMPK (p-AMPK) were determined by real-time fluorescence quantitation of PCRQ RT-PCRR). The mRNA expression of chemokine ligand 2 CCL 2 and ICAM 1) was observed. Results the protein expression of NF 魏 B p65: compared with the control group, the protein expression of p65 was significantly increased in the PA group, and decreased significantly in the Met PA combination group compared with the PA group, especially in the Met PA combined group compared with the other Met PA combination groups. Compared with the control group, the protein expression of p-I 魏 B 伪 increased significantly in the PA group, and decreased significantly in the Met PA combined group compared with the PA group, especially in the Met PA combined group compared with the other concentration groups, and the protein expression level in the Met PA group was significantly lower than that in the other concentration group (P < 0.05). The protein expression level in the white cells was lower than that in the control group, and that in the Met PA group was significantly lower than that in the control group. The expression of PA combined with histone protein was lower than that of the control group (P < 0.05). The expression of ICAM1 was significantly higher in the PA group than in the control group, and decreased with the increase of the Met concentration gradient in the PA group, and the protein table of P0.05U. 4p-AMPK was decreased with the increase of the concentration gradient of Met. Volume: compared with the control group and PA group, the cells of Met PA combined group increased with the increase of Met concentration gradient. The protein expression in Met PA group was significantly higher than that in other concentrations of Met PA combined group, and the mRNA expression of ICAM1 was higher in Met PA combination group than in other concentrations. The mRNA expression of ICAM1 in Met PA group was significantly higher than that in control group, and that in Met PA group was higher than that in PA group, and that in PA group was higher than that in PA group. The expression level of CCL2 decreased with the increase of Met concentration gradient in the combination group. Compared with the control group, the expression level of CCL2 in the Met PA combined group increased significantly (P 0.05), and the expression level in the Met PA combined group decreased with the increase of the Met concentration gradient in the PA group (P < 0.05), but the expression level in the Met PA combined group decreased with the increase of the Met concentration gradient (P < 0.05), while that in the control group was significantly higher than that in the control group (P < 0.05). Conclusion: metformin can attenuate the inflammatory response of vascular endothelial cells induced by palmitate by inhibiting NF 魏 B pathway, and decrease the expression of adhesion factor and chemokine induced by palmitic acid. In endothelial cells supplemented with metformin, adenylate activated protein kinase was activated by phosphorylation, and its activation may be related to the inactivation of NF 魏 B pathway.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1;R54

【参考文献】