PPAR-γ激动剂Pioglitazone在血管平滑肌细胞中调节KLF4表达的分子机制研究

发布时间：2018-05-31 21:57

  本文选题：血管平滑肌细胞 + Pioglitazone　； 参考：《河北医科大学》2013年硕士论文

【摘要】：目的：Krüppel样因子4（krüppel-like factor4, KLF4）是一种含有锌指结构的转录因子,其C端含有3个连续的C2H2锌指结构。KLF4通过直接或间接与靶基因启动子区DNA相结合，来调控相关靶基因的表达。已经证明KLF4调节细胞增殖、分化、发育、凋亡和炎症等细胞生物学行为。在血管平滑肌细胞（vascular smooth muscle cell, VSMC)中，KLF4通过调节VSMC标志基因和细胞周期调节基因的表达，对VSMC表型转化发挥重要的调节作用。 过氧化物酶体增殖物激活受体（peroxisome proliferator-activatedreceptors, PPARs）是一类配体激活的核转录因子，可以通过直接或间接与目的基因启动子DNA相结合，来调控相关靶基因的表达。PPARs有三种亚型，即α、β/δ和γ。其中，对PPAR-γ的研究最为深入。PPAR-γ在多种细胞中均表达，包括脂肪细胞、巨噬细胞、血管内皮细胞（endothelial cell,EC）和VSMC等。PPAR-γ能抑制细胞增殖、促进细胞分化并具有抗炎及抗动脉粥样硬化等作用。Pioglitazone作为PPAR-γ的激动剂，已经广泛应用于动脉粥样硬化、心肌梗死等多种心血管疾病的治疗。 KLF4和PPAR-γ在调节细胞增殖和分化之间的关系是人们感兴趣的问题。然而，在VSMC中，PPAR-γ是否能调控KLF4的表达尚不清楚。本研究观察PPAR-γ激动剂Pioglitazone在血管平滑肌细胞中对KLF4表达的影响，探讨PPAR-γ调控KLF4表达的分子机制。 方法：Western blot分析检测Pioglitazone对KLF4表达及不同信号通路活化的影响；转染靶向PPAR-γ的小干扰RNA敲低内源性PPAR-γ表达，检测KLF4蛋白表达；Real-time PCR检测KLF4mRNA水平；用荧光素酶报告基因分析检测Pioglitazone和PPAR-γ对KLF4基因转录的影响；细胞免疫荧光染色分析检测KLF4的表达量。 结果： 1PPAR-γ激动剂Pioglitazone促进KLF4蛋白表达 Western blot分析显示，随着Pioglitazone浓度增加，KLF4的表达水平呈剂量依赖性升高，于10μΜ时达到高峰。随着Pioglitazone刺激时间的延长，KLF4的表达呈时间依赖性增加，于刺激12h后开始增加，刺激24h达高峰。细胞免疫荧光染色结果也显示，，与对照组相比，Pioglitazone刺激后，KLF4免疫荧光染色强度呈时间和剂量依赖性增加。这些结果表明, Pioglitazone促进KLF4的表达。 2Pioglitazone以依赖于其受体PPAR-γ的方式促进KLF4表达 为了研究Pioglitazone是否通过激活其受体PPAR-γ来调控KLF4表达，我们用靶向PPAR-γ的siRNA转染VSMC，检测Pioglitazone对KLF4表达的影响。Western blot分析结果显示，敲低PPAR-γ表达后，KLF4蛋白表达水平降低；此外，敲低PPAR-γ表达能取消Pioglitazone对KLF4表达的促进作用。这些结果表明，PPAR-γ激动剂Pioglitazone诱导KLF4的表达依赖于其受体PPAR-γ。 3PPAR-γ激动剂Pioglitazone不影响KLF4基因转录 上述结果表明，PPAR-γ激动剂Pioglitazone促进KLF4蛋白表达。为了检测其对KLF4mRNA表达水平有无影响，我们进行了real-time PCR分析。结果发现，Pioglitazone对KLF4mRNA水平没有明显影响。荧光素酶报告基因分析结果显示，PPAR-γ表达质粒FLAG-PPAR-γ转染NIH-3T3细胞后， KLF4基因启动子活性没有明显变化，同时给予Pioglitazone刺激亦不影响KLF4基因启动子活性。上述结果表明，PPAR-γ激动剂Pioglitazone不影响KLF4基因转录。 4PPAR-γ激动剂Pioglitazone增强KLF4蛋白的稳定性 上述结果显示，PPAR-γ激动剂Pioglitazone促进KLF4蛋白表达，但不影响KLF4mRNA表达水平。我们推测，Pioglitazone能增强KLF4蛋白的稳定性。用20μg/ml放线菌酮（cycloheximide, CHX）孵育VSMC,Western blot检测KLF4蛋白水平，结果显示，用CHX阻断KLF4蛋白的从头合成后，Pioglitazone处理能使KLF4蛋白保持较高水平。上述结果表明，PPAR-γ激动剂Pioglitazone能增强KLF4蛋白的稳定性。 5PPAR-γ激动剂Pioglitazone通过激活Akt信号通路增强KLF4蛋白的稳定性 为进一步探讨Pioglitazone是通过激活哪条信号通路来增强KLF4蛋白的稳定性，我们首先检测了Pioglitazone对ERK、p38MAPK和Akt信号通路的影响。Pioglitazone（10μΜ）刺激VSMC0.5h，p38和Akt的磷酸化水平均显著升高,而ERK的磷酸化水平没有明显变化。结果说明，Pioglitazone能激活p38和Akt信号通路。我们用不同信号通路的抑制剂预孵育VSMC2h后，再给予Pioglitazone刺激24h，收集细胞进行Westernblot分析。结果表明，用ly294002抑制Akt信号通路后，Pioglitazone不再增加KLF4蛋白水平，即失去对KLF4蛋白的稳定作用。给予SB431542和PD98059后，Pioglitazone仍然能增加KLF4蛋白水平。这些结果表明，Pioglitazone通过激活Akt信号转导通路增强KLF4蛋白稳定性。 结论： 1PPAR-γ激动剂Pioglitazone促进KLF4蛋白表达。 2Pioglitazone以依赖于其受体PPAR-γ的方式促进KLF4表达。 3PPAR-γ激动剂Pioglitazone不影响KLF4基因转录。 4PPAR-γ激动剂Pioglitazone增强KLF4蛋白的稳定性。 5PPAR-γ激动剂Pioglitazone通过激活Akt信号通路增强KLF4蛋白的稳定性。
[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.
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