本文選題：內(nèi)皮通透性 + 內(nèi)脂素　； 參考：《南華大學》2011年碩士論文

【摘要】：背景與目的：Visfatin稱之為內(nèi)脂素，它是一種來源于內(nèi)臟脂肪的新脂肪因子,具有復雜的生物學功能，除具有類似胰島素的降血糖效應外,還可能作為炎癥介質(zhì)參與體內(nèi)炎癥反應。大量證據(jù)表明內(nèi)脂素可能在心腦血管疾病、糖尿病、代謝綜合征和腎臟疾病等動脈粥樣硬化性疾病中發(fā)揮著重要作用，這與內(nèi)脂素影響斑塊穩(wěn)定性，血管炎癥及糖脂代謝等作用密切相關(guān)。近年來，研究認為它可能是一個潛在危險因素參與了內(nèi)皮功能紊亂的發(fā)生。然而，內(nèi)脂素對血管內(nèi)皮功能的影響復雜，到目前為止，一直沒有直接證據(jù)證實內(nèi)脂素能導致動脈內(nèi)皮損傷和闡明其作用機制。本研究從內(nèi)皮細胞間連接角度出發(fā)，，擬觀察內(nèi)脂素對人臍靜脈內(nèi)皮細胞通透性及縫隙連接相關(guān)蛋白Connexin37、Connexin40和Connexin43表達的影響及初步探討其可能機制。 方法：將HUVEC-12以5x104/孔的密度種板，經(jīng)不同濃度（0、1、10、100nM）和不同時間（0、3、6、12、24h）visfatin處理，或預先Wortmannin (50nM)、U0126(50μM)、SB203580(50μM)等抑制劑孵育3h。運用實時熒光定量PCR和Western印跡分析法分別檢測Connexin37、Connexin40和Connexin43mRNA與蛋白質(zhì)水平的變化，并采用transwell system檢測單層內(nèi)皮通透性的改變。 結(jié)果：Visfatin處理內(nèi)皮細胞對Cx37、Cx40mRNA表達無影響，但Cx37、Cx40蛋白的表達隨著visfatin濃度的增高和時間延長而減少，呈一定劑量、時間依賴性。然而，visfatin能呈劑量、時間依賴性上調(diào)Cx43mRNA及蛋白的表達。通透性實驗結(jié)果顯示隨著visfatin濃度的增加及處理時間的延長，內(nèi)皮細胞致密單層的通透性逐漸增大，且該作用亦呈劑量和時間依賴性。此外，Wortmannin特異性抑制PI3K活性后，能明顯逆轉(zhuǎn)visfatin誘導的Cx43蛋白表達，但對Cx43的mRNA的表達無影響。U0126特異性抑制ERK1/2活性或SB203580特異性抑制p38MAPK活性則均能明顯下調(diào)visfatin誘導的Cx43mRNA及蛋白表達。 結(jié)論： 1、visfatin可下調(diào)內(nèi)皮細胞Cx37、Cx40蛋白表達，上調(diào)Cx43mRNA和蛋白的表達 2、visfatin可增加單層內(nèi)皮的通透性，且呈時間、劑量依賴性。 3、PI3K、ERK1/2及p38MAPK可介導visfatin誘導HUVEC-12Cx43的表達。
[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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

【參考文獻】

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1 陳敏;蔣麗萍;洪濤;;縫隙連接蛋白在動脈粥樣硬化形成和防治中的作用[J];中國藥理學通報;2010年10期

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