本文關(guān)鍵詞：Krüppel樣因子4在實(shí)驗(yàn)性肺纖維化中的表達(dá)及干預(yù)研究，由筆耕文化傳播整理發(fā)布。

        目的：特發(fā)性肺纖維化（idiopathic pulmonary fibrosis, IPF）是一種病因不明的慢性間質(zhì)性肺疾病，其發(fā)病機(jī)制尚未明確，以早期的肺泡炎癥和晚期的纖維增生為主要特征。他汀類藥物是一類3－羥基－3－甲基戊二酰輔酶A（3-hydroxy-3-methylglutaryl-coenzyme A, HMG-CoA）還原酶抑制劑，通過(guò)對(duì)HMG-CoA還原酶的抑制作用，使血清膽固醇合成減少。他汀類藥物除降脂作用外，還具有抗炎、抗氧化、抗血小板凝集、改善內(nèi)皮功能等作用，既往研究已表明他汀類的抗炎作用可改善肺臟、肝臟、腎臟等的纖維化，但其作用機(jī)制尚不明確。Krüppel樣因子4（Krüppel-like factor4，KLF4）是在多種組織中廣泛表達(dá)的、具有雙重功能的轉(zhuǎn)錄因子，在細(xì)胞的生長(zhǎng)、增殖、分化和凋亡等眾多生理活動(dòng)中起著重要作用。近年來(lái)研究發(fā)現(xiàn)，KLF4與心血管疾病、腫瘤和炎性疾病等均有密切關(guān)系，而KLF4在炎癥反應(yīng)中的作用主要表現(xiàn)為對(duì)單核巨噬細(xì)胞表型偏移的調(diào)節(jié)。為了探討KLF4在阿托伐他汀抑制實(shí)驗(yàn)性肺纖維化中的作用，本研究擬通過(guò)支氣管滴注法建立小鼠肺纖維化模型，以觀察KLF4基因和蛋白在肺纖維化小鼠肺組織中的表達(dá)，通過(guò)阿托伐他汀干預(yù)肺纖維化小鼠，觀察阿托伐他汀干預(yù)后肺纖維化小鼠肺組織中KLF4的表達(dá)變化，并通過(guò)RNA干擾技術(shù)敲除巨噬細(xì)胞RAW264.7KLF4基因，觀察KLF4對(duì)巨噬細(xì)胞細(xì)胞增殖、凋亡及細(xì)胞表型的影響，進(jìn)一步探討KLF4在阿托伐他汀改善實(shí)驗(yàn)性肺纖維化過(guò)程中的作用機(jī)制。第一部分KLF4在實(shí)驗(yàn)性肺纖維化小鼠肺組織中的動(dòng)態(tài)表達(dá)方法：1C57BL/6小鼠隨機(jī)分為生理鹽水對(duì)照組（Control）和博萊霉素組(BLM)，，經(jīng)氣管內(nèi)注入博萊霉素(2.5mg/kg)建立實(shí)驗(yàn)性小鼠肺纖維化模型,經(jīng)氣管內(nèi)注入等量生理鹽水作為對(duì)照組，分別于術(shù)后第12h、1d、2d、3d、7d、14d、28d取材；2采用HE染色和Masson染色觀察肺組織病理變化及膠原的沉積部位和數(shù)量；3實(shí)時(shí)定量PCR法檢測(cè)各組肺組織中KLF4mRNA表達(dá)水平；4免疫組織化學(xué)法檢測(cè)各組肺組織中KLF4蛋白表達(dá)水平。結(jié)果：1博萊霉素誘導(dǎo)的小鼠肺纖維化過(guò)程中，第1、2、3天表現(xiàn)為急性炎癥，第7天炎癥加重，并出現(xiàn)膠原沉積，第14天肺泡結(jié)構(gòu)塌陷，膠原沉積明顯，第28天肺泡結(jié)構(gòu)基本正常，炎癥程度和膠原沉積較輕；2與對(duì)照組相比，BLM組小鼠肺組織中KLF4mRNA的表達(dá)水平第1天開(kāi)始升高，而后降低，第3天達(dá)最低后逐漸升高直至第28天。3與對(duì)照組相比，BLM組小鼠肺組織中KLF4蛋白的表達(dá)水平與mRNA表達(dá)趨勢(shì)基本一致，第1天開(kāi)始升高，而后降低，第3天達(dá)最低后逐漸升高至第14天后再次下降。第二部分阿托伐他汀對(duì)實(shí)驗(yàn)性肺纖維化小鼠肺組織中KLF4表達(dá)變化的影響方法：1將C57BL/6小鼠隨機(jī)分為對(duì)照組（Control）、博萊霉素組（BLM）和阿托伐他汀組（ATO），BLM組和ATO組一次性經(jīng)氣管內(nèi)注入博萊霉素(2.5mg/kg)建立實(shí)驗(yàn)性小鼠肺纖維化模型,對(duì)照組注入等量生理鹽水。ATO組在氣管滴注BLM后給予阿托伐他汀灌胃10mg/(kg·d)，分別于術(shù)后第3d、14d、28d取材；2采用HE染色、Masson染色的方法觀察肺組織病理變化及膠原的沉積部位和數(shù)量；3實(shí)時(shí)定量PCR法檢測(cè)各組肺組織中KLF4mRNA表達(dá)水平；4免疫組織化學(xué)法檢測(cè)各組肺組織中KLF4蛋白表達(dá)水平；5明膠酶譜分析法檢測(cè)各組肺組織基質(zhì)金屬蛋白酶-2（matrixmetalloproteinase-2, MMP-2）的活性。結(jié)果：1病理結(jié)果顯示ATO組較BLM組炎癥程度和膠原沉積明顯減輕。2與BLM組（0.336±0.195）相比，ATO組（2.050±1.564）KLF4mRNA表達(dá)水平第3天明顯上調(diào)，差異有統(tǒng)計(jì)學(xué)意義（P<0.05），第14天ATO組（0.464±0.110）較BLM組（2.000±0.435）明顯下調(diào)，差異有統(tǒng)計(jì)學(xué)意義（P<0.05）。3與BLM組相比，各時(shí)間點(diǎn)ATO組KLF4蛋白表達(dá)水平均下調(diào),差異有統(tǒng)計(jì)學(xué)意義（P<0.05）。4BLM組MMP-2活性較對(duì)照組明顯上調(diào)，阿托伐他汀干預(yù)后MMP-2的活性明顯下調(diào)，差異有統(tǒng)計(jì)學(xué)意義（P<0.05）。第三部分KLF4對(duì)巨噬細(xì)胞細(xì)胞增殖、凋亡及表型偏移的影響方法：1通過(guò)LipofectamineTM2000將短發(fā)夾RNA（short hairpin，shRNA）干擾質(zhì)粒轉(zhuǎn)染至RAW264.7，經(jīng)G418篩選獲得穩(wěn)定細(xì)胞株，獲得的穩(wěn)定干擾KLF4的RAW264.7細(xì)胞株命名為shKLF4，表達(dá)對(duì)照質(zhì)粒的細(xì)胞株命名為NC，野生型RAW264.7細(xì)胞命名為WT;2實(shí)時(shí)定量PCR法（RT-PCR）和Western-blot法鑒定干擾效率；3RT-PCR法檢測(cè)巨噬細(xì)胞表型偏移：獲得穩(wěn)定干擾KLF4細(xì)胞株后，脂多糖LPS誘導(dǎo)巨噬細(xì)胞表型偏移，用RT-PCR法檢測(cè)巨噬細(xì)胞表型標(biāo)志物的相對(duì)表達(dá)量；4CCK-8法測(cè)定穩(wěn)定細(xì)胞株的增殖活性；5流式細(xì)胞術(shù)檢測(cè)巨噬細(xì)胞的細(xì)胞周期及細(xì)胞凋亡；結(jié)果：1成功建立穩(wěn)定干擾KLF4的細(xì)胞株，RT-PCR法和Western-blot法鑒定干擾效率達(dá)70%以上，；2RT-PCR法結(jié)果顯示，LPS誘導(dǎo)后，抑制巨噬細(xì)胞KLF4的表達(dá)使M1型巨噬細(xì)胞標(biāo)志物TNF-α，MCP-1及CCL5的表達(dá)降低（P<0.05）；3CCK-8法結(jié)果顯示，降低巨噬細(xì)胞KLF4的表達(dá)使細(xì)胞增殖活性下降（P<0.05）；4流式細(xì)胞術(shù)結(jié)果顯示，抑制巨噬細(xì)胞KLF4的表達(dá)促進(jìn)巨噬細(xì)胞的凋亡（P<0.05）；結(jié)論：1KLF4在實(shí)驗(yàn)性肺纖維化發(fā)生發(fā)展中呈現(xiàn)明顯的動(dòng)態(tài)變化；2阿托伐他汀干預(yù)博萊霉素致肺纖維化小鼠后KLF4表達(dá)明顯下調(diào)；3KLF4低表達(dá)對(duì)巨噬細(xì)胞的細(xì)胞周期無(wú)明顯影響，但促進(jìn)其凋亡，抑制其增殖，同時(shí)抑制巨噬細(xì)胞向M1型偏移；4阿托伐他汀可能通過(guò)抑制KLF4的表達(dá)來(lái)實(shí)現(xiàn)其在實(shí)驗(yàn)性肺纖維化中的抗炎和抗纖維化作用。

    Objective Idiopathic pulmonary fibrosis (IPF) is a chronic lung diseaseof unknown cause characterized by alveolitis in the early stage and fibrosis inthe advanced stage, the understanding of it’s pathogenesis remains unkown.Statins are3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductaseinhibitors, which are used to lower serum cholesterol synthesis through theinhibition of HMG-CoA reductase. Besides lipid-lowering, statins also has theeffects of anti-inflammation, antioxidant, anti-platelet aggregation andimproving endothelial function. Previous research has shown that theanti-inflammatory effects of statins can attenuate the fibrosis in the organs likelung, liver, kidney and so on, but its mechanism is still not clear. Krüppel-likefactor4is a transcription factor, with dual function, it is expressed in manytissues, and plays an important role in cell proliferation, differentiation andapoptosis in many physiological activities. Recent studies found thatcardiovascular disease, cancer and inflammatory diseases are closedly relatedto KLF4, and the role of KLF4in the inflammation is mainly manifested bythe regulation of monocyte and macrophage phenotype shifting. To investigatethe effect of KLF4on the inhibition of pulmomary fibrosis through thetreatment of atorvastatin, this study aimed to establish a mouse model ofpulmonary fibrosis by intratracheal instillation of bleomycin, to observe theexpression of KLF4gene and protein in the lung tissues of mice; to give theintervation of atorvastatin in mice with pulmonary fibrosis, to observe thechange of KLF4expression in the lung tissues of mice; and to acquire the cellline with stable KLF4gene silencing through RNA interference (RNAi) inmurine RAW264.7macrophages, to investigate the influence of KLF4inproliferation, apoptosis and the phnotype in this cell line, and then furtherexplore the mechanism of KLF4in inhibition of atorvastatin in experimental pulmonary fibrosis.Part one Dynamic Expression of Krüppel-like factor4in the LungTissues of Mice with Pulmonary FibrosisMethods:1C57BL/6mice were randomly divided into Control group and BLMgroup. Mice in the BLM group were given a single intratracheal injection ofbleomycin (2.5mg/kg), while those in the Control group were injected withisodose physiological saline. Groups were sacrificed on the hour12and theday1,2,3,7,14and28;2Hematoxylin and eosin stain （HE stain） and Masson’s TrichromeStain were used to detect the pathology of alveolar and the deposition ofcellularity and collagen;3Real time-polymerase chain reaction (RT-PCR) was performed toinvestigate the expression of KLF4gene in the lung tissues of mice;4Immunohistochemical technology was performed to investigate theexpression of KLF4protein in the lung tissues of mice.Results:1In the bleomycin-induced pulmonary fibrosis, acute inflammation wasperformed on the day1,2and3, and the inflammation was exacerbated andthe collagen deposition began to be observed on the day7, the architecture ofalveolar was destroyed and the collagen deposition was more obvious on theday14, while the alveolar structure was nearly recovered to normal and theinflammation and collagen deposition were attenuated on the day28;2The expression of KLF4mRNA increased from the day1, thendecreased, arrived at the minimum on the day3, and then gradually increaseduntil the day28;3The trend of KLF4protein showed roughly the same as the KLF4mRNA level, it started to increase on the day1, then decreased, arrived at theminimum on the day3, then gradually increased until the day14and thendecreased again.Part two The Effect of Atorvastatin on the Expression of KLF4in the Lung Tissues of Mice with Pulmonary FibrosisMethods:1C57BL/6were randomly divided into3groups: Control group,bleomycin group (BLM) and atorvastatin group (ATO). Group BLM and ATOwere given a single intratracheal injection of bleomycin (2.5mg/kg), whilegroup Control was injected with isodose physiological saline. Group ATO wastreated with atorvastatin10mg/(kg d) by intragastric administration the dayafter bleomycin instillation. All groups were sacrificed on the day3,14and28;2Hematoxylin and eosin stain （HE stain） and Masson’s TrichromeStain were used to detect the pathology of alveolar and the deposition ofcellularity and collagen;3Real time-polymerase chain reaction (RT-PCR) was performed toinvestigate the expression of KLF4gene in the lung tissues of mice;4Immunohistochemical technology was performed to investigate theexpression of KLF4protein in the lung tissues of mice;5Zymography was used to investigate the activation of matrixmetalloproteinase-2（MMP-2）.Results:1Pathological results showed that compared with the BLM group, theinflammation and the collagen deposition were significantly reduced in theATO group;2Compared with BLM group（0.336±0.195）, the expression of KLF4mRNA significantly（P<0.05） increased in the ATO group（2.050±1.564）on the day3, and the expression of KLF4mRNA significantly（P<0.05）decreased in the ATO group（0.464±0.110）compared with BLM group（2.000±0.435） on the day14;3Compared with BLM group, the expression of KLF4proteinsignificantly （P<0.05）decreased in the ATO group on each time point;4The activation of MMP-2significantly（P<0.05）increased in the groupBLM compared with the Control group, and significantly （P<0.05） decreased after the treatment of atorvastatin.Part three Effects of KLF4on Cell proliferation, Apoptosis andPhenotype shifting in macrophagesMethods:1KLF4deficient stable cell line was generated by selection in G418afterthe transfection of short hairpin (shRNA) plasmid with LipofectamineTM2000.The obtained cell line KLF4deficiency was named shKLF4, the controlplasmid expression cell line was named NC, wild type RAW264.7cells wasnamed WT;2RNAi efficiency was qualified by real-time quantitative polymerasechain reaction (RT-PCR) and Western-blot;3Macrophage phenotype shifting was detected by RT-PCR:After KLF4deficient stable cell line generated, LPS was used to induce the macrophageporlarization, and RT-PCR was used to detect the relative expression of M1macrophage biomarkers;4Proliferation ability was measured by CCK-8;5Cell cycle and apoptosis were analyzed by flow cytometry;Results:1Stable KLF4-deficient cell line was established and the expression ofKLF4was reduced by70%;2After LPS induced, the expression of M1macrophages biomarkerTNF-α，MCP-1and CCL5decreased in KLF4deficient stable cell line;3Decreased proliferation was observed in KLF4deficient stable cell lineby CCK-8(P<0.05);4Lower expression of KLF4in macrophages promote cell apoptosisthrough flow cytometry (P<0.05).Conclusion:1The expression of KLF4is dynamically changed in the process ofexperimental pulmonary fibrosis;2Ater the intervention of atorvastatin, the expression of KLF4isobviously decreased in the experimental pulmonary fibrosis; 3The cell cycle in the RAW264.7macrophages with KLF4lower-expression is not obviously different from the wide type macrophages.However, KLF4lower-expression could suppress the proliferation andpromote apoptosis, and inhibit macrophage M1polarization;4KLF4may be the mediator in the effects of anti-inflammation andanti-fibrosis of atorvastatin in the expremental pulmonary fibrosis.

Krüppel樣因子4在實(shí)驗(yàn)性肺纖維化中的表達(dá)及干預(yù)研究

摘要5-9ABSTRACT9-13引言14-17    參考文獻(xiàn)15-17第一部分 KLF4 在實(shí)驗(yàn)性肺纖維化小鼠肺組織中的動(dòng)態(tài)表達(dá)17-31    前言17    材料與方法17-23    結(jié)果23-24    附圖24-27    附表27-28    討論28-29    小結(jié)29    參考文獻(xiàn)29-31第二部分 阿托伐他汀對(duì)實(shí)驗(yàn)性肺纖維化小鼠肺組織中 KLF4 表達(dá)的影響31-44    前言31    材料與方法31-34    結(jié)果34-36    附圖36-40    附表40-41    討論41-42    小結(jié)42    參考文獻(xiàn)42-44第三部分 干擾 KLF4 對(duì) RAW264.7 巨噬細(xì)胞細(xì)胞增殖、凋亡及表型偏移的影響44-57    前言44    材料與方法44-49    結(jié)果49-51    附圖51-54    附表54-55    討論55-56    小結(jié)56    參考文獻(xiàn)56-57結(jié)論57-58綜述 Krüppel 樣因子 4 在肺纖維化中的作用58-65    參考文獻(xiàn)61-65致謝65-66個(gè)人簡(jiǎn)歷66

  本文關(guān)鍵詞：Krüppel樣因子4在實(shí)驗(yàn)性肺纖維化中的表達(dá)及干預(yù)研究，由筆耕文化傳播整理發(fā)布。