本文選題：二甲雙胍 + 內(nèi)皮細(xì)胞�。� 參考：《貴州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:本實驗探討二甲雙胍(Metformin,Met)對軟脂酸(palmitic acid,PA)誘導(dǎo)損傷的血管內(nèi)皮細(xì)胞是否具有保護作用,并對其可能發(fā)生的機制做進一步研究,為糖尿病合并心血管病患者的治療提供新思路和靶點。方法:(1)將人臍靜脈內(nèi)皮細(xì)胞(human umbilical vein endothelial cells,HUVEC)分為5組,依次為空白對照組(常規(guī)細(xì)胞培養(yǎng))、PA組(300μmol/L)、低濃度Met(1mmol/L)+PA聯(lián)合組、中濃度Met(2mmol/L)+PA聯(lián)合組、高濃度Met(4mmol/L)+PA聯(lián)合組,在無血清培養(yǎng)液中培養(yǎng)24小時后用CCK-8法測定各組細(xì)胞活性。(2)用蛋白質(zhì)印跡法(Westenblot)測定各組細(xì)胞核因子κB(nuclear factorκB p65,NFκB p65)、細(xì)胞間黏附因子(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)的蛋白表達(dá)。(3)用實時熒光定量PCR(q RT-PCR)的方法測定各組細(xì)胞單核細(xì)胞趨化因子(Chemokine ligand 2,CCL2)和細(xì)胞間黏附因子(ICAM1)的mRNA表達(dá)。結(jié)果:(1)NFκB p65的蛋白表達(dá)量:與對照組相比,PA組表達(dá)量明顯升高(P0.05);與PA組相比,Met+PA聯(lián)合組細(xì)胞表達(dá)量明顯降低(P均0.05),其中中濃度Met+PA聯(lián)合組較其他濃度Met+PA聯(lián)合組蛋白表達(dá)量更低(P均0.05)。(2)p-IκBα的蛋白表達(dá)量:與對照組相比,PA組表達(dá)量明顯升高(P0.05);與PA組相比,Met+PA聯(lián)合組細(xì)胞表達(dá)量明顯降低(P均0.05),其中中濃度Met+PA聯(lián)合組較其他濃度Met+PA聯(lián)合組蛋白表達(dá)量更低(P均0.05)。(3)ICAM1的蛋白表達(dá)量:與對照組相比,PA組表達(dá)量明顯升高(P0.05);與PA組相比,Met+PA聯(lián)合組細(xì)胞表達(dá)量隨Met濃度梯度增高而遞減(P均0.05)。(4)p-AMPK的蛋白表達(dá)量:與對照組和PA組相比,Met+PA聯(lián)合組細(xì)胞隨著Met濃度梯度的增加,其蛋白表達(dá)量顯著增高(P均0.05),其中中濃度Met+PA聯(lián)合組較其他濃度Met+PA聯(lián)合組蛋白表達(dá)量更高(P均0.05)。(5)ICAM1的mRNA表達(dá)量:與對照組相比,PA組表達(dá)量有所增加(P0.05);與PA組相比,Met+PA聯(lián)合組表達(dá)量隨Met濃度梯度的升高而減少(P均0.05)。(6)CCL2的mRNA表達(dá)量:與對照組相比,PA組表達(dá)量顯著增加(P0.05);與PA組相比,Met+PA聯(lián)合組表達(dá)量隨Met濃度梯度的升高而減少(P均0.05)。結(jié)論:二甲雙胍可以通過抑制NFκB通路減輕軟脂酸誘導(dǎo)的血管內(nèi)皮細(xì)胞炎性反應(yīng),降低軟脂酸在細(xì)胞內(nèi)引起的細(xì)胞粘附因子和趨化因子的表達(dá),對細(xì)胞起到一定的保護作用;而在加入二甲雙胍的內(nèi)皮細(xì)胞中,腺苷酸活化蛋白激酶被磷酸化激活,它的激活可能與NFκB通路的失活有重要聯(lián)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 (VECs) 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 偽, phosphorylated nuclear factor- 魏 B inhibitor protein p-I 魏 B 偽, 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 偽 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.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1;R54

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