本文選題：血管生成素樣蛋白4(Angptl4) + 過氧化氫(H_2O_2)�。� 參考：《山東大學(xué)》2017年碩士論文

【摘要】：血管生成素樣蛋白4(Angiopoietin-like protein 4,Angptpl4)自被發(fā)現(xiàn)以來,其在疾病中的作用得到越來越多的研究和重視�，F(xiàn)已有研究表明Angpt14參與動脈粥樣硬化的發(fā)生發(fā)展。但是其確切的作用機(jī)制仍不清楚。同樣地,大量的研究表明氧化應(yīng)激與心血管疾病密切相關(guān),但其發(fā)揮作用的途徑還需要進(jìn)一步探究。隨著研究的深入,氧化應(yīng)激與炎癥反應(yīng)在動脈粥樣硬疾病中所起的作用進(jìn)一步被揭示,已經(jīng)成為探究其發(fā)病機(jī)制及尋找新的治療手段的研究重點。研究目的:本實驗旨在探究過氧化氫(hydrogen peroxide,H_2O_2)對小鼠巨噬細(xì)胞株RAW264.7中血管生成素樣蛋白4(Angptl4)水平的影響以及進(jìn)一步探究參與H_2O_2調(diào)節(jié)Angpt14表達(dá)的可能機(jī)制。研究方法:1.選用小鼠來源的巨噬細(xì)胞株RAW264.7;2.將巨噬細(xì)胞接種至細(xì)胞培養(yǎng)板,待細(xì)胞穩(wěn)定并且貼壁,將細(xì)胞分為12組。正常對照組,H_2O_2 刺激組(0.25mmol/L),H_2O_2 刺激組(0.5mmol/L),H_2O_2刺激組(0.25mmol/L)+ U0126(20mmol/L),H_2O_2刺激組(0.5mmol/L)+U0126(20mmol/L),U0126 抑制組(20mmol/L),H_2O_2刺激組(0.25mmol/L)+ SB203580(40mmol/L),H_2O_2刺激組(0.5mmol/L)+SB203580(40mmol/L),SB203580 抑制組(40mmol/L),H_2O_2 刺激組(0.25mmol/L)+SP600125(10mmol/L),H_2O_2刺激組(0.5mmol/L)+SP600125(10mmol/L),SP600125抑制組(10mmol/L)。3.收集各組細(xì)胞并提取蛋白;收集各組培養(yǎng)基。應(yīng)用細(xì)胞免疫熒光法、Western Blot技術(shù)分別檢測各組細(xì)胞中Angptl4的表達(dá);應(yīng)用ELISA法檢測各組培養(yǎng)基中Angpt4的水平;應(yīng)用Western Blot技術(shù)檢測MAPKs通路磷酸化蛋白的表達(dá)量。實驗結(jié)果:1.H_2O_2促進(jìn)巨噬細(xì)胞RAW264.7中Angptl4的表達(dá):研究發(fā)現(xiàn)不同濃度(0.25mmol/L、0.5mmol/L)的 H_2O_2刺激 RAW264.7 細(xì)胞 24 小時,可以促進(jìn)巨噬細(xì)胞中Angpt14的表達(dá),并且呈濃度依賴性(P0.05)。2.H_2O_2激活巨噬細(xì)胞RAW264.7中MAPKs通路:研究發(fā)現(xiàn)給予RAW264.7細(xì)胞 H_2O_2(0.25mmol/L、0.5mmol/L)刺激 24h,細(xì)胞中的磷酸化 ERKl/2,p38 MAPK以及JNK通路蛋白表達(dá)均增加,且呈濃度依賴性(P0.05)。3.ERK1/2,p38MAPK通路拮抗劑抑制H_2O_2對Angpt14表達(dá)的影響:分別在U0126(20mmol/L)和 SB203580(40mmol/L)預(yù)處理 90 分鐘條件下,H_2O_2(0.25mmol/L、0.5mmol/L)+ U0126(20mmol/L)組和 H_2O_2(0.25mmol/L、0.5mmol/L)+ SB203580(20mmol/L)組中的 Angptl4 的表達(dá)量均較單純H_2O_2刺激組(0.25mmol/L、0.5mmol/L)組明顯減少(P0.05),而在 SP600125(lOmmol/L)預(yù)處理 30 分鐘條件下,H_2O_2(0.25mmol/L、0.5mmol/L)+SP600125(10mmol/L)組和 H_2O_2刺激組(0.25mmol/L、0.5mmol/L)組相比無明顯統(tǒng)計學(xué)差異(P0.05)。結(jié)論及意義:1.H_2O_2可促進(jìn)巨噬細(xì)胞中Angpt14的表達(dá),并呈濃度依賴性。2.H_2O_2可激活巨噬細(xì)胞中的MAPKs通路,其中ERK1/2和p38 MAPK通路在H_2O_2調(diào)控的Angpt14表達(dá)中發(fā)揮關(guān)鍵作用。
[Abstract]:Since its discovery, the role of angiopoietin-like protein (4(Angiopoietin-like protein _ 4 / pAngtpl4) in disease has received more and more attention. Studies have shown that Angpt14 is involved in the development of atherosclerosis. However, the exact mechanism of its action is still unclear. Similarly, a large number of studies have shown that oxidative stress is closely related to cardiovascular disease, but the ways in which it works need to be further explored. With the development of the research, the role of oxidative stress and inflammation in atherosclerosis has been revealed further, which has become the focus of research on the pathogenesis of atherosclerosis and the search for new treatment methods. Objective: to investigate the effect of hydrogen peroxide-H _ tid _ 2O _ 2 (H _ 2O _ 2) on the level of angiopoietin like protein (4Angptl4) in mouse macrophage cell line RAW264.7 and the possible mechanism involved in the regulation of Angpt14 expression by H_2O_2. Research method: 1. Mouse macrophage cell line RAW264.7 was selected. The macrophages were inoculated into the cell culture plate and the cells were stable and adhered to the wall. The cells were divided into 12 groups. 姝ｅ父瀵圭収緇，

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