脂联素对高糖诱导血管内皮细胞LOX-1表达的调控作用研究

发布时间：2018-01-24 01:33

本文关键词： D-葡萄糖人脐静脉内皮细胞脂联素 LOX-1 NF－κB ERK1/2　出处：《南华大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：研究脂肪细胞因子脂联素（APN）对高糖诱导血管内皮细胞凝集素样氧化型低密度脂蛋白受体-1（LOX-1）表达的影响及其机制，为2型糖尿病患者大血管病变的预防和治疗提供理论依据。方法：（1）不同浓度D-葡萄糖(0、15、30、60mmol/L)作用于人脐静脉内皮细胞株（HUVEC-12）48h后，倒置相差显微镜观察内皮细胞形态，Western Blotting检测HUVEC-12中LOX-1的蛋白表达，筛选最佳葡萄糖浓度用于构建高糖诱导的内皮细胞受损模型。（2）用浓度30mmol/LD-葡萄糖作用于HUVEC-12不同时间（0、12、24、48、72h）后，倒置相差显微镜观察内皮细胞形态，Western Blotting检测HUVEC-12中LOX-1的蛋白表达，筛选最佳葡萄糖作用时间用于建模。（3）选取30mmol/L D-葡萄糖作用于HUVEC-1248h，造成D-葡萄糖诱导的HUVEC-12内皮功能受损模型；然后，不同浓度的APN（0、10、20、40μg/m l）作用于D-葡萄糖诱导的HUVEC-1248h，倒置相差显微镜观察内皮细胞形态，Western Blotting检测磷酸化细胞外信号调节激酶1/2(p-ERK1/2)、核转录因子（NF）－κB和LOX-1蛋白表达，同时，筛选最佳保护作用的APN浓度。（4）选取20μg/m l APN作用于D-葡萄糖诱导的HUVEC-12不同时间（0、12、24、48、72h）后，倒置相差显微镜观察内皮细胞形态，Western Blotting检测HUVEC-12中p-ERK1/2、NF－κB和LOX-1蛋白表达。同时，筛选最佳保护作用的APN作用时间。（5）在20μg/ml APN作用48h后的D-葡萄糖诱导的HUVEC-12中使用ERK1/2阻断剂PD98059（50umol/l）干预，用Western Blotting检测HUVEC-12中p-ERK1/2、NF－κB和LOX-1蛋白表达,用间接免疫荧光法检测其核转录因子－κB亚基p65（NF－κB-p65）核转位情况。结果：（1）不同浓度的D-葡萄糖诱导HUVEC-12不同时间，镜下见内皮细胞排列较为紊乱,折光性变弱，细胞核模糊，细胞间隙明显增大，细胞内颗粒状物增多；D-葡萄糖诱导的HUVEC-12中LOX-1蛋白表达均上调，其中，30mmol/L D-葡萄糖作用于HUVEC-1248h，内皮细胞形态改变和LOX-1蛋白表达上调最明显。（2）不同浓度APN作用于D-葡萄糖诱导的HUVEC-12不同时间，镜下见内皮细胞形态改善，折光性有所增强，胞内颗粒物减少，细胞排列相对规整；同时，APN可使D-葡萄糖诱导的HUVEC-12内LOX-1蛋白表达下调，，NF－κB表达降低，ERK1/2磷酸化水平增强；其中20μg/ml APN作用于D-葡萄糖诱导的HUVEC-1248h后，作用最明显。（3）ERK阻断剂PD98059和APN联合干预下的D-葡萄糖诱导的HUVEC-12中ERK信号通路受抑制，LOX-1蛋白表达上调， NF－κB-p65蛋白表达增多，并向细胞核内转移增多，出现核移位。结论： 1．高糖可诱使HUVEC-12形态出现受损性改变，并可诱使HUVEC-12的LOX-1蛋白表达上调。 2． APN可能通过激活ERK1/2信号通路，引起NF－κB蛋白表达下调，从而引起D-葡萄糖诱导的HUVEC-12中LOX-1蛋白表达下调，发挥其保护血管内皮细胞的作用。
[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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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