本文關(guān)鍵詞： D-葡萄糖 人臍靜脈內(nèi)皮細(xì)胞 脂聯(lián)素 LOX-1 NF－κB ERK1/2　出處：《南華大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：研究脂肪細(xì)胞因子脂聯(lián)素（APN）對高糖誘導(dǎo)血管內(nèi)皮細(xì)胞凝集素樣氧化型低密度脂蛋白受體-1（LOX-1）表達(dá)的影響及其機(jī)制，為2型糖尿病患者大血管病變的預(yù)防和治療提供理論依據(jù)。 方法： （1）不同濃度D-葡萄糖(0、15、30、60mmol/L)作用于人臍靜脈內(nèi)皮細(xì)胞株（HUVEC-12）48h后，倒置相差顯微鏡觀察內(nèi)皮細(xì)胞形態(tài)，Western Blotting檢測HUVEC-12中LOX-1的蛋白表達(dá)，篩選最佳葡萄糖濃度用于構(gòu)建高糖誘導(dǎo)的內(nèi)皮細(xì)胞受損模型。 （2）用濃度30mmol/LD-葡萄糖作用于HUVEC-12不同時間（0、12、24、48、72h）后，倒置相差顯微鏡觀察內(nèi)皮細(xì)胞形態(tài)，Western Blotting檢測HUVEC-12中LOX-1的蛋白表達(dá)，篩選最佳葡萄糖作用時間用于建模。 （3）選取30mmol/L D-葡萄糖作用于HUVEC-1248h，造成D-葡萄糖誘導(dǎo)的HUVEC-12內(nèi)皮功能受損模型；然后，不同濃度的APN（0、10、20、40μg/m l）作用于D-葡萄糖誘導(dǎo)的HUVEC-1248h，倒置相差顯微鏡觀察內(nèi)皮細(xì)胞形態(tài)，Western Blotting檢測磷酸化細(xì)胞外信號調(diào)節(jié)激酶1/2(p-ERK1/2)、核轉(zhuǎn)錄因子（NF）－κB和LOX-1蛋白表達(dá)，同時，篩選最佳保護(hù)作用的APN濃度。 （4）選取20μg/m l APN作用于D-葡萄糖誘導(dǎo)的HUVEC-12不同時間（0、12、24、48、72h）后，倒置相差顯微鏡觀察內(nèi)皮細(xì)胞形態(tài)，Western Blotting檢測HUVEC-12中p-ERK1/2、NF－κB和LOX-1蛋白表達(dá)。同時，篩選最佳保護(hù)作用的APN作用時間。 （5）在20μg/ml APN作用48h后的D-葡萄糖誘導(dǎo)的HUVEC-12中使用ERK1/2阻斷劑PD98059（50umol/l）干預(yù)，用Western Blotting檢測HUVEC-12中p-ERK1/2、NF－κB和LOX-1蛋白表達(dá),用間接免疫熒光法檢測其核轉(zhuǎn)錄因子－κB亞基p65（NF－κB-p65）核轉(zhuǎn)位情況。 結(jié)果： （1）不同濃度的D-葡萄糖誘導(dǎo)HUVEC-12不同時間，鏡下見內(nèi)皮細(xì)胞排列較為紊亂,折光性變?nèi)�，�?xì)胞核模糊，細(xì)胞間隙明顯增大，細(xì)胞內(nèi)顆粒狀物增多；D-葡萄糖誘導(dǎo)的HUVEC-12中LOX-1蛋白表達(dá)均上調(diào)，其中，30mmol/L D-葡萄糖作用于HUVEC-1248h，內(nèi)皮細(xì)胞形態(tài)改變和LOX-1蛋白表達(dá)上調(diào)最明顯。 （2）不同濃度APN作用于D-葡萄糖誘導(dǎo)的HUVEC-12不同時間，鏡下見內(nèi)皮細(xì)胞形態(tài)改善，折光性有所增強(qiáng)，胞內(nèi)顆粒物減少，細(xì)胞排列相對規(guī)整；同時，APN可使D-葡萄糖誘導(dǎo)的HUVEC-12內(nèi)LOX-1蛋白表達(dá)下調(diào)，，NF－κB表達(dá)降低，ERK1/2磷酸化水平增強(qiáng)；其中20μg/ml APN作用于D-葡萄糖誘導(dǎo)的HUVEC-1248h后，作用最明顯。 （3）ERK阻斷劑PD98059和APN聯(lián)合干預(yù)下的D-葡萄糖誘導(dǎo)的HUVEC-12中ERK信號通路受抑制，LOX-1蛋白表達(dá)上調(diào)， NF－κB-p65蛋白表達(dá)增多，并向細(xì)胞核內(nèi)轉(zhuǎn)移增多，出現(xiàn)核移位。 結(jié)論： 1．高糖可誘使HUVEC-12形態(tài)出現(xiàn)受損性改變，并可誘使HUVEC-12的LOX-1蛋白表達(dá)上調(diào)。 2． APN可能通過激活ERK1/2信號通路，引起NF－κB蛋白表達(dá)下調(diào)，從而引起D-葡萄糖誘導(dǎo)的HUVEC-12中LOX-1蛋白表達(dá)下調(diào)，發(fā)揮其保護(hù)血管內(nèi)皮細(xì)胞的作用。
[Abstract]:Objective: to study the effect of adiponectin (APN) on the expression of lectin like oxidized low density lipoprotein receptor (LOX-1) in vascular endothelial cells induced by high glucose and its mechanism. To provide theoretical basis for the prevention and treatment of macrovascular disease in type 2 diabetes mellitus. Methods: (1) 60 mmol / L of different concentrations of D-glucosamine (D- 1) on human umbilical vein endothelial cell line HUVEC-1210 for 48 h. The expression of LOX-1 protein in HUVEC-12 was detected by reverse phase contrast microscope and Western Blotting. The best glucose concentration was selected to construct the model of endothelial cell damage induced by high glucose. (2) the endothelial cells were observed by inverted phase contrast microscope after treated with 30 mmol / L LD- glucose at different time points in HUVEC-12 for 48 ~ 72 h. The expression of LOX-1 protein in HUVEC-12 was detected by Western Blotting, and the optimal glucose action time was selected for modeling. (3) 30 mmol / L D- glucose was applied to HUVEC-1248 h to induce impaired endothelial function of HUVEC-12 induced by D-glucose. Then, the HUVEC-induced HUVEC-1248h was treated with different concentrations of APNN010 / L 20g / ml, and the morphology of endothelial cells was observed by inverted phase contrast microscope. Western Blotting was used to detect the expression of phosphorylated extracellular signal-regulated kinase (1 / 2) -ERK / 1 / 2, nuclear transcription factor (NF)-魏 B and LOX-1 protein. Screening the best concentration of APN for protection. (4) 20 渭 g / ml APN was used to treat HUVEC-12 induced by D-glucose at different time. Reverse phase contrast microscope was used to observe the morphology of endothelial cells. Western Blotting was used to detect p-ERK1 / 2 in HUVEC-12. NF- 魏 B and LOX-1 protein expression. At the same time, screening the best protective effect of APN action time. 5) use of ERK1/2 blocker PD98059550umol / L in HUVEC-12 induced by D-glucose at 20 渭 g / ml APN for 48 h). Intervention. Western Blotting was used to detect the expression of NF- 魏 B and LOX-1 protein in HUVEC-12. The nuclear translocation of nuclear transcription factor-魏 B subunit p65-魏 B-p65 was detected by indirect immunofluorescence assay. Results: 1) at different concentrations of D-glucose induced HUVEC-12 at different time, the endothelial cells arranged in disorder, refractive index weakened, nucleus blurred and intercellular gap enlarged obviously under microscope. The number of intracellular particles increased. The expression of LOX-1 protein was up-regulated in HUVEC-12 induced by D-glucose, and 30 mmol / L D- glucose acted on HUVEC-1248h. Morphological changes of endothelial cells and up-regulation of LOX-1 protein expression were most obvious. (2) when different concentrations of APN were applied to HUVEC-12 induced by D-glucose for different time, the morphology of endothelial cells was improved, the refractive index was enhanced, and the intracellular particulate matter was decreased. Cell arrangement was relatively regular; At the same time, the expression of LOX-1 protein in HUVEC-12 induced by D-glucose decreased the expression of NF- 魏 B and increased the phosphorylation level of ERK1 / 2. The effect of 20 渭 g / ml APN on HUVEC-1248h induced by D-glucose was the most obvious. The ERK signaling pathway in HUVEC-12 induced by D- glucose induced by PD98059 and APN inhibited the up-regulation of ERK-1 protein expression. The expression of NF- 魏 B-p65 protein was increased, and the nuclear translocation was observed. Conclusion: 1. High glucose could induce damage to the morphology of HUVEC-12 and up-regulate the expression of LOX-1 protein in HUVEC-12. 2. APN may induce down-regulation of NF- 魏 B protein by activating ERK1/2 signaling pathway. The expression of LOX-1 protein in HUVEC-12 induced by D-glucose was down-regulated, which could protect vascular endothelial cells.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

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