本文選題：血管平滑肌細胞 + Pioglitazone�。� 參考：《河北醫(yī)科大學》2013年碩士論文

【摘要】：目的：Krüppel樣因子4（krüppel-like factor4, KLF4）是一種含有鋅指結構的轉錄因子,其C端含有3個連續(xù)的C2H2鋅指結構。KLF4通過直接或間接與靶基因啟動子區(qū)DNA相結合，來調控相關靶基因的表達。已經(jīng)證明KLF4調節(jié)細胞增殖、分化、發(fā)育、凋亡和炎癥等細胞生物學行為。在血管平滑肌細胞（vascular smooth muscle cell, VSMC)中，KLF4通過調節(jié)VSMC標志基因和細胞周期調節(jié)基因的表達，對VSMC表型轉化發(fā)揮重要的調節(jié)作用。 過氧化物酶體增殖物激活受體（peroxisome proliferator-activatedreceptors, PPARs）是一類配體激活的核轉錄因子，可以通過直接或間接與目的基因啟動子DNA相結合，來調控相關靶基因的表達。PPARs有三種亞型，即α、β/δ和γ。其中，對PPAR-γ的研究最為深入。PPAR-γ在多種細胞中均表達，包括脂肪細胞、巨噬細胞、血管內皮細胞（endothelial cell,EC）和VSMC等。PPAR-γ能抑制細胞增殖、促進細胞分化并具有抗炎及抗動脈粥樣硬化等作用。Pioglitazone作為PPAR-γ的激動劑，已經(jīng)廣泛應用于動脈粥樣硬化、心肌梗死等多種心血管疾病的治療。 KLF4和PPAR-γ在調節(jié)細胞增殖和分化之間的關系是人們感興趣的問題。然而，在VSMC中，PPAR-γ是否能調控KLF4的表達尚不清楚。本研究觀察PPAR-γ激動劑Pioglitazone在血管平滑肌細胞中對KLF4表達的影響，探討PPAR-γ調控KLF4表達的分子機制。 方法：Western blot分析檢測Pioglitazone對KLF4表達及不同信號通路活化的影響；轉染靶向PPAR-γ的小干擾RNA敲低內源性PPAR-γ表達，檢測KLF4蛋白表達；Real-time PCR檢測KLF4mRNA水平；用熒光素酶報告基因分析檢測Pioglitazone和PPAR-γ對KLF4基因轉錄的影響；細胞免疫熒光染色分析檢測KLF4的表達量。 結果： 1PPAR-γ激動劑Pioglitazone促進KLF4蛋白表達 Western blot分析顯示，隨著Pioglitazone濃度增加，KLF4的表達水平呈劑量依賴性升高，于10μΜ時達到高峰。隨著Pioglitazone刺激時間的延長，KLF4的表達呈時間依賴性增加，于刺激12h后開始增加，刺激24h達高峰。細胞免疫熒光染色結果也顯示，，與對照組相比，Pioglitazone刺激后，KLF4免疫熒光染色強度呈時間和劑量依賴性增加。這些結果表明, Pioglitazone促進KLF4的表達。 2Pioglitazone以依賴于其受體PPAR-γ的方式促進KLF4表達 為了研究Pioglitazone是否通過激活其受體PPAR-γ來調控KLF4表達，我們用靶向PPAR-γ的siRNA轉染VSMC，檢測Pioglitazone對KLF4表達的影響。Western blot分析結果顯示，敲低PPAR-γ表達后，KLF4蛋白表達水平降低；此外，敲低PPAR-γ表達能取消Pioglitazone對KLF4表達的促進作用。這些結果表明，PPAR-γ激動劑Pioglitazone誘導KLF4的表達依賴于其受體PPAR-γ。 3PPAR-γ激動劑Pioglitazone不影響KLF4基因轉錄 上述結果表明，PPAR-γ激動劑Pioglitazone促進KLF4蛋白表達。為了檢測其對KLF4mRNA表達水平有無影響，我們進行了real-time PCR分析。結果發(fā)現(xiàn)，Pioglitazone對KLF4mRNA水平?jīng)]有明顯影響。熒光素酶報告基因分析結果顯示，PPAR-γ表達質粒FLAG-PPAR-γ轉染NIH-3T3細胞后， KLF4基因啟動子活性沒有明顯變化，同時給予Pioglitazone刺激亦不影響KLF4基因啟動子活性。上述結果表明，PPAR-γ激動劑Pioglitazone不影響KLF4基因轉錄。 4PPAR-γ激動劑Pioglitazone增強KLF4蛋白的穩(wěn)定性 上述結果顯示，PPAR-γ激動劑Pioglitazone促進KLF4蛋白表達，但不影響KLF4mRNA表達水平。我們推測，Pioglitazone能增強KLF4蛋白的穩(wěn)定性。用20μg/ml放線菌酮（cycloheximide, CHX）孵育VSMC,Western blot檢測KLF4蛋白水平，結果顯示，用CHX阻斷KLF4蛋白的從頭合成后，Pioglitazone處理能使KLF4蛋白保持較高水平。上述結果表明，PPAR-γ激動劑Pioglitazone能增強KLF4蛋白的穩(wěn)定性。 5PPAR-γ激動劑Pioglitazone通過激活Akt信號通路增強KLF4蛋白的穩(wěn)定性 為進一步探討Pioglitazone是通過激活哪條信號通路來增強KLF4蛋白的穩(wěn)定性，我們首先檢測了Pioglitazone對ERK、p38MAPK和Akt信號通路的影響。Pioglitazone（10μΜ）刺激VSMC0.5h，p38和Akt的磷酸化水平均顯著升高,而ERK的磷酸化水平?jīng)]有明顯變化。結果說明，Pioglitazone能激活p38和Akt信號通路。我們用不同信號通路的抑制劑預孵育VSMC2h后，再給予Pioglitazone刺激24h，收集細胞進行Westernblot分析。結果表明，用ly294002抑制Akt信號通路后，Pioglitazone不再增加KLF4蛋白水平，即失去對KLF4蛋白的穩(wěn)定作用。給予SB431542和PD98059后，Pioglitazone仍然能增加KLF4蛋白水平。這些結果表明，Pioglitazone通過激活Akt信號轉導通路增強KLF4蛋白穩(wěn)定性。 結論： 1PPAR-γ激動劑Pioglitazone促進KLF4蛋白表達。 2Pioglitazone以依賴于其受體PPAR-γ的方式促進KLF4表達。 3PPAR-γ激動劑Pioglitazone不影響KLF4基因轉錄。 4PPAR-γ激動劑Pioglitazone增強KLF4蛋白的穩(wěn)定性。 5PPAR-γ激動劑Pioglitazone通過激活Akt信號通路增強KLF4蛋白的穩(wěn)定性。
[Abstract]:Objective: Kr u ppel like factor 4 (KR u ppel-like factor4, KLF4) is a transcription factor containing zinc finger structure. Its C terminal contains 3 consecutive C2H2 zinc finger structures that regulate the expression of related target genes by direct or indirect combination with DNA of the target gene promoter region. It has been shown that KLF4 regulates cell proliferation, differentiation, development, apoptosis and inflammation. In vascular smooth muscle cells (vascular smooth muscle cell, VSMC), KLF4 plays an important role in regulating the phenotype transformation of VSMC by regulating the expression of the VSMC marker gene and the cell cycle regulating gene.
The peroxisome proliferator activated receptor (peroxisome proliferator-activatedreceptors, PPARs) is a class of ligand activated nuclear transcription factors, which can be combined directly or indirectly with the target gene promoter DNA to regulate the expression of related target genes in three subtypes, namely, alpha, beta / delta and gamma. Among them, the study of PPAR- gamma is the most important. .PPAR- gamma is expressed in a variety of cells, including adipocytes, macrophages, vascular endothelial cells (endothelial cell, EC) and VSMC, and.PPAR- gamma can inhibit cell proliferation, promote cell differentiation and have anti-inflammatory and anti atherosclerosis effects.Pioglitazone as an agonist of PPAR- gamma, which has been widely used in atherosclerosis, Treatment of a variety of cardiovascular diseases, such as myocardial infarction.
The relationship between KLF4 and PPAR- gamma in regulating cell proliferation and differentiation is an issue of interest. However, it is not clear whether PPAR- gamma can regulate the expression of KLF4 in VSMC. This study observed the effect of PPAR- gamma agonist Pioglitazone on the expression of KLF4 in vascular smooth muscle cells and explored the molecular mechanism of PPAR- gamma regulation of the expression of KLF4.
Methods: Western blot analysis was used to detect the effect of Pioglitazone on the expression of KLF4 and the activation of different signal pathways, and the small interference RNA transfected to PPAR- gamma knocked down the expression of endogenous PPAR- gamma, detected the expression of KLF4 protein, and the Real-time PCR detected the KLF4mRNA level, and the luciferase reporter gene was used to analyze the gene transfer of Pioglitazone and gamma. The expression of KLF4 was detected by cellular immunofluorescence staining.
Result錛

本文編號：1961410