本文選題：血管平滑肌細(xì)胞 + Pioglitazone　； 參考：《河北醫(yī)科大學(xué)》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(jié)構(gòu)的轉(zhuǎn)錄因子,其C端含有3個(gè)連續(xù)的C2H2鋅指結(jié)構(gòu)。KLF4通過(guò)直接或間接與靶基因啟動(dòng)子區(qū)DNA相結(jié)合，來(lái)調(diào)控相關(guān)靶基因的表達(dá)。已經(jīng)證明KLF4調(diào)節(jié)細(xì)胞增殖、分化、發(fā)育、凋亡和炎癥等細(xì)胞生物學(xué)行為。在血管平滑肌細(xì)胞（vascular smooth muscle cell, VSMC)中，KLF4通過(guò)調(diào)節(jié)VSMC標(biāo)志基因和細(xì)胞周期調(diào)節(jié)基因的表達(dá)，對(duì)VSMC表型轉(zhuǎn)化發(fā)揮重要的調(diào)節(jié)作用。 過(guò)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(zhuǎn)錄因子，可以通過(guò)直接或間接與目的基因啟動(dòng)子DNA相結(jié)合，來(lái)調(diào)控相關(guān)靶基因的表達(dá)。PPARs有三種亞型，即α、β/δ和γ。其中，對(duì)PPAR-γ的研究最為深入。PPAR-γ在多種細(xì)胞中均表達(dá)，包括脂肪細(xì)胞、巨噬細(xì)胞、血管內(nèi)皮細(xì)胞（endothelial cell,EC）和VSMC等。PPAR-γ能抑制細(xì)胞增殖、促進(jìn)細(xì)胞分化并具有抗炎及抗動(dòng)脈粥樣硬化等作用。Pioglitazone作為PPAR-γ的激動(dòng)劑，已經(jīng)廣泛應(yīng)用于動(dòng)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(diào)節(jié)細(xì)胞增殖和分化之間的關(guān)系是人們感興趣的問(wèn)題。然而，在VSMC中，PPAR-γ是否能調(diào)控KLF4的表達(dá)尚不清楚。本研究觀察PPAR-γ激動(dòng)劑Pioglitazone在血管平滑肌細(xì)胞中對(duì)KLF4表達(dá)的影響，探討PPAR-γ調(diào)控KLF4表達(dá)的分子機(jī)制。 方法：Western blot分析檢測(cè)Pioglitazone對(duì)KLF4表達(dá)及不同信號(hào)通路活化的影響；轉(zhuǎn)染靶向PPAR-γ的小干擾RNA敲低內(nèi)源性PPAR-γ表達(dá)，檢測(cè)KLF4蛋白表達(dá)；Real-time PCR檢測(cè)KLF4mRNA水平；用熒光素酶報(bào)告基因分析檢測(cè)Pioglitazone和PPAR-γ對(duì)KLF4基因轉(zhuǎn)錄的影響；細(xì)胞免疫熒光染色分析檢測(cè)KLF4的表達(dá)量。 結(jié)果： 1PPAR-γ激動(dòng)劑Pioglitazone促進(jìn)KLF4蛋白表達(dá) Western blot分析顯示，隨著Pioglitazone濃度增加，KLF4的表達(dá)水平呈劑量依賴性升高，于10μΜ時(shí)達(dá)到高峰。隨著Pioglitazone刺激時(shí)間的延長(zhǎng)，KLF4的表達(dá)呈時(shí)間依賴性增加，于刺激12h后開(kāi)始增加，刺激24h達(dá)高峰。細(xì)胞免疫熒光染色結(jié)果也顯示，，與對(duì)照組相比，Pioglitazone刺激后，KLF4免疫熒光染色強(qiáng)度呈時(shí)間和劑量依賴性增加。這些結(jié)果表明, Pioglitazone促進(jìn)KLF4的表達(dá)。 2Pioglitazone以依賴于其受體PPAR-γ的方式促進(jìn)KLF4表達(dá) 為了研究Pioglitazone是否通過(guò)激活其受體PPAR-γ來(lái)調(diào)控KLF4表達(dá)，我們用靶向PPAR-γ的siRNA轉(zhuǎn)染VSMC，檢測(cè)Pioglitazone對(duì)KLF4表達(dá)的影響。Western blot分析結(jié)果顯示，敲低PPAR-γ表達(dá)后，KLF4蛋白表達(dá)水平降低；此外，敲低PPAR-γ表達(dá)能取消Pioglitazone對(duì)KLF4表達(dá)的促進(jìn)作用。這些結(jié)果表明，PPAR-γ激動(dòng)劑Pioglitazone誘導(dǎo)KLF4的表達(dá)依賴于其受體PPAR-γ。 3PPAR-γ激動(dòng)劑Pioglitazone不影響KLF4基因轉(zhuǎn)錄 上述結(jié)果表明，PPAR-γ激動(dòng)劑Pioglitazone促進(jìn)KLF4蛋白表達(dá)。為了檢測(cè)其對(duì)KLF4mRNA表達(dá)水平有無(wú)影響，我們進(jìn)行了real-time PCR分析。結(jié)果發(fā)現(xiàn)，Pioglitazone對(duì)KLF4mRNA水平?jīng)]有明顯影響。熒光素酶報(bào)告基因分析結(jié)果顯示，PPAR-γ表達(dá)質(zhì)粒FLAG-PPAR-γ轉(zhuǎn)染NIH-3T3細(xì)胞后， KLF4基因啟動(dòng)子活性沒(méi)有明顯變化，同時(shí)給予Pioglitazone刺激亦不影響KLF4基因啟動(dòng)子活性。上述結(jié)果表明，PPAR-γ激動(dòng)劑Pioglitazone不影響KLF4基因轉(zhuǎn)錄。 4PPAR-γ激動(dòng)劑Pioglitazone增強(qiáng)KLF4蛋白的穩(wěn)定性 上述結(jié)果顯示，PPAR-γ激動(dòng)劑Pioglitazone促進(jìn)KLF4蛋白表達(dá)，但不影響KLF4mRNA表達(dá)水平。我們推測(cè)，Pioglitazone能增強(qiáng)KLF4蛋白的穩(wěn)定性。用20μg/ml放線菌酮（cycloheximide, CHX）孵育VSMC,Western blot檢測(cè)KLF4蛋白水平，結(jié)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(jié)果表明，PPAR-γ激動(dòng)劑Pioglitazone能增強(qiáng)KLF4蛋白的穩(wěn)定性。 5PPAR-γ激動(dòng)劑Pioglitazone通過(guò)激活A(yù)kt信號(hào)通路增強(qiáng)KLF4蛋白的穩(wěn)定性 為進(jìn)一步探討Pioglitazone是通過(guò)激活哪條信號(hào)通路來(lái)增強(qiáng)KLF4蛋白的穩(wěn)定性，我們首先檢測(cè)了Pioglitazone對(duì)ERK、p38MAPK和Akt信號(hào)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(jié)果說(shuō)明，Pioglitazone能激活p38和Akt信號(hào)通路。我們用不同信號(hào)通路的抑制劑預(yù)孵育VSMC2h后，再給予Pioglitazone刺激24h，收集細(xì)胞進(jìn)行Westernblot分析。結(jié)果表明，用ly294002抑制Akt信號(hào)通路后，Pioglitazone不再增加KLF4蛋白水平，即失去對(duì)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(jié)果表明，Pioglitazone通過(guò)激活A(yù)kt信號(hào)轉(zhuǎn)導(dǎo)通路增強(qiáng)KLF4蛋白穩(wěn)定性。 結(jié)論： 1PPAR-γ激動(dòng)劑Pioglitazone促進(jìn)KLF4蛋白表達(dá)。 2Pioglitazone以依賴于其受體PPAR-γ的方式促進(jìn)KLF4表達(dá)。 3PPAR-γ激動(dòng)劑Pioglitazone不影響KLF4基因轉(zhuǎn)錄。 4PPAR-γ激動(dòng)劑Pioglitazone增強(qiáng)KLF4蛋白的穩(wěn)定性。 5PPAR-γ激動(dòng)劑Pioglitazone通過(guò)激活A(yù)kt信號(hào)通路增強(qiáng)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(hào)：1961410