脂肪因子Visfatin对人脐静脉单层内皮通透性的影响及机制初步探讨

发布时间：2018-07-06 14:47

本文选题：内皮通透性 + 内脂素　；参考：《南华大学》2011年硕士论文

【摘要】：背景与目的：Visfatin称之为内脂素，它是一种来源于内脏脂肪的新脂肪因子,具有复杂的生物学功能，除具有类似胰岛素的降血糖效应外,还可能作为炎症介质参与体内炎症反应。大量证据表明内脂素可能在心脑血管疾病、糖尿病、代谢综合征和肾脏疾病等动脉粥样硬化性疾病中发挥着重要作用，这与内脂素影响斑块稳定性，血管炎症及糖脂代谢等作用密切相关。近年来，研究认为它可能是一个潜在危险因素参与了内皮功能紊乱的发生。然而，内脂素对血管内皮功能的影响复杂，到目前为止，一直没有直接证据证实内脂素能导致动脉内皮损伤和阐明其作用机制。本研究从内皮细胞间连接角度出发，，拟观察内脂素对人脐静脉内皮细胞通透性及缝隙连接相关蛋白Connexin37、Connexin40和Connexin43表达的影响及初步探讨其可能机制。方法：将HUVEC-12以5x104/孔的密度种板，经不同浓度（0、1、10、100nM）和不同时间（0、3、6、12、24h）visfatin处理，或预先Wortmannin (50nM)、U0126(50μM)、SB203580(50μM)等抑制剂孵育3h。运用实时荧光定量PCR和Western印迹分析法分别检测Connexin37、Connexin40和Connexin43mRNA与蛋白质水平的变化，并采用transwell system检测单层内皮通透性的改变。结果：Visfatin处理内皮细胞对Cx37、Cx40mRNA表达无影响，但Cx37、Cx40蛋白的表达随着visfatin浓度的增高和时间延长而减少，呈一定剂量、时间依赖性。然而，visfatin能呈剂量、时间依赖性上调Cx43mRNA及蛋白的表达。通透性实验结果显示随着visfatin浓度的增加及处理时间的延长，内皮细胞致密单层的通透性逐渐增大，且该作用亦呈剂量和时间依赖性。此外，Wortmannin特异性抑制PI3K活性后，能明显逆转visfatin诱导的Cx43蛋白表达，但对Cx43的mRNA的表达无影响。U0126特异性抑制ERK1/2活性或SB203580特异性抑制p38MAPK活性则均能明显下调visfatin诱导的Cx43mRNA及蛋白表达。结论： 1、visfatin可下调内皮细胞Cx37、Cx40蛋白表达，上调Cx43mRNA和蛋白的表达 2、visfatin可增加单层内皮的通透性，且呈时间、剂量依赖性。 3、PI3K、ERK1/2及p38MAPK可介导visfatin诱导HUVEC-12Cx43的表达。
[Abstract]:Background & AIM: visfatin is a new adipose factor derived from visceral fat, which has complex biological functions. In addition to the hypoglycemic effect similar to insulin, visfatin may also participate in the inflammatory response as an inflammatory mediator in vivo. Evidence suggests that endolipin may play an important role in atherosclerotic diseases such as cardio-cerebrovascular disease, diabetes, metabolic syndrome and kidney disease, which may affect plaque stability. Vascular inflammation and glucose and lipid metabolism are closely related. In recent years, it has been suggested that it may be a potential risk factor for endothelial dysfunction. However, the effects of endolipin on vascular endothelial function are complex. So far, there has been no direct evidence that endolipin can cause arterial endothelial injury and clarify its mechanism. From the point of view of endothelial cell junctions, this study was designed to investigate the effect of endolipin on the permeability of human umbilical vein endothelial cells and the expression of gap junction associated proteins Connexin37, Connexin40 and Connexin43, and to explore its possible mechanism. Methods: HUVEC-12 was incubated with 5x104/ pore density seed plate at different concentrations (01C 10100nM) and at different time (0210NM) for 24 h, or pre-incubated with Wortmannin (50nm) U0126 (50 渭 M) SB203580 (50 渭 M) for 3 h. The levels of Connexin 40 and Connexin 43 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blotting, respectively, and the changes of endothelial permeability of monolayer were detected by transwell system. Results the expression of Cx37 and Cx40 mRNA in endothelial cells was not affected by w: Visfatin treatment, but the expression of Cx37 Cx40 protein decreased with the increase of visfatin concentration and time, in a dose-dependent and time-dependent manner. However, visfatin could up-regulate the expression of Cx43 mRNA and protein in a dose and time dependent manner. The results of permeability experiment showed that the permeability of dense monolayer of endothelial cells increased gradually with the increase of visfatin concentration and treatment time, and the effect was in a dose-and time-dependent manner. In addition, Wortmannin specifically inhibited PI3K activity and reversed the expression of Cx43 protein induced by visfatin. However, U0126 specifically inhibited ERK1 / 2 activity or SB203580 specifically inhibited p38MAPK activity. However, U0126 specifically inhibited Cx43 mRNA and protein expression induced by visfatin. Conclusion: 1. Visfatin can down-regulate the expression of Cx37 and Cx40 protein in endothelial cells, and upregulate the expression of Cx43 mRNA and protein. 2. Visfatin can increase the permeability of endothelial monolayer. In a dose-dependent manner, the expression of HUVEC-12Cx43 was mediated by p38 MAPK and PI3K ERK1 / 2.
【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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