Krüppel样因子4在实验性肺纤维化中的表达及干预研究

  本文关键词：Krüppel样因子4在实验性肺纤维化中的表达及干预研究，由笔耕文化传播整理发布。

        目的：特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）是一种病因不明的慢性间质性肺疾病，其发病机制尚未明确，以早期的肺泡炎症和晚期的纤维增生为主要特征。他汀类药物是一类3－羟基－3－甲基戊二酰辅酶A（3-hydroxy-3-methylglutaryl-coenzyme A, HMG-CoA）还原酶抑制剂，通过对HMG-CoA还原酶的抑制作用，使血清胆固醇合成减少。他汀类药物除降脂作用外，还具有抗炎、抗氧化、抗血小板凝集、改善内皮功能等作用，既往研究已表明他汀类的抗炎作用可改善肺脏、肝脏、肾脏等的纤维化，但其作用机制尚不明确。Krüppel样因子4（Krüppel-like factor4，KLF4）是在多种组织中广泛表达的、具有双重功能的转录因子，在细胞的生长、增殖、分化和凋亡等众多生理活动中起着重要作用。近年来研究发现，KLF4与心血管疾病、肿瘤和炎性疾病等均有密切关系，而KLF4在炎症反应中的作用主要表现为对单核巨噬细胞表型偏移的调节。为了探讨KLF4在阿托伐他汀抑制实验性肺纤维化中的作用，本研究拟通过支气管滴注法建立小鼠肺纤维化模型，以观察KLF4基因和蛋白在肺纤维化小鼠肺组织中的表达，通过阿托伐他汀干预肺纤维化小鼠，观察阿托伐他汀干预后肺纤维化小鼠肺组织中KLF4的表达变化，并通过RNA干扰技术敲除巨噬细胞RAW264.7KLF4基因，观察KLF4对巨噬细胞细胞增殖、凋亡及细胞表型的影响，进一步探讨KLF4在阿托伐他汀改善实验性肺纤维化过程中的作用机制。第一部分KLF4在实验性肺纤维化小鼠肺组织中的动态表达方法：1C57BL/6小鼠随机分为生理盐水对照组（Control）和博莱霉素组(BLM)，，经气管内注入博莱霉素(2.5mg/kg)建立实验性小鼠肺纤维化模型,经气管内注入等量生理盐水作为对照组，分别于术后第12h、1d、2d、3d、7d、14d、28d取材；2采用HE染色和Masson染色观察肺组织病理变化及胶原的沉积部位和数量；3实时定量PCR法检测各组肺组织中KLF4mRNA表达水平；4免疫组织化学法检测各组肺组织中KLF4蛋白表达水平。结果：1博莱霉素诱导的小鼠肺纤维化过程中，第1、2、3天表现为急性炎症，第7天炎症加重，并出现胶原沉积，第14天肺泡结构塌陷，胶原沉积明显，第28天肺泡结构基本正常，炎症程度和胶原沉积较轻；2与对照组相比，BLM组小鼠肺组织中KLF4mRNA的表达水平第1天开始升高，而后降低，第3天达最低后逐渐升高直至第28天。3与对照组相比，BLM组小鼠肺组织中KLF4蛋白的表达水平与mRNA表达趋势基本一致，第1天开始升高，而后降低，第3天达最低后逐渐升高至第14天后再次下降。第二部分阿托伐他汀对实验性肺纤维化小鼠肺组织中KLF4表达变化的影响方法：1将C57BL/6小鼠随机分为对照组（Control）、博莱霉素组（BLM）和阿托伐他汀组（ATO），BLM组和ATO组一次性经气管内注入博莱霉素(2.5mg/kg)建立实验性小鼠肺纤维化模型,对照组注入等量生理盐水。ATO组在气管滴注BLM后给予阿托伐他汀灌胃10mg/(kg·d)，分别于术后第3d、14d、28d取材；2采用HE染色、Masson染色的方法观察肺组织病理变化及胶原的沉积部位和数量；3实时定量PCR法检测各组肺组织中KLF4mRNA表达水平；4免疫组织化学法检测各组肺组织中KLF4蛋白表达水平；5明胶酶谱分析法检测各组肺组织基质金属蛋白酶-2（matrixmetalloproteinase-2, MMP-2）的活性。结果：1病理结果显示ATO组较BLM组炎症程度和胶原沉积明显减轻。2与BLM组（0.336±0.195）相比，ATO组（2.050±1.564）KLF4mRNA表达水平第3天明显上调，差异有统计学意义（P<0.05），第14天ATO组（0.464±0.110）较BLM组（2.000±0.435）明显下调，差异有统计学意义（P<0.05）。3与BLM组相比，各时间点ATO组KLF4蛋白表达水平均下调,差异有统计学意义（P<0.05）。4BLM组MMP-2活性较对照组明显上调，阿托伐他汀干预后MMP-2的活性明显下调，差异有统计学意义（P<0.05）。第三部分KLF4对巨噬细胞细胞增殖、凋亡及表型偏移的影响方法：1通过LipofectamineTM2000将短发夹RNA（short hairpin，shRNA）干扰质粒转染至RAW264.7，经G418筛选获得稳定细胞株，获得的稳定干扰KLF4的RAW264.7细胞株命名为shKLF4，表达对照质粒的细胞株命名为NC，野生型RAW264.7细胞命名为WT;2实时定量PCR法（RT-PCR）和Western-blot法鉴定干扰效率；3RT-PCR法检测巨噬细胞表型偏移：获得稳定干扰KLF4细胞株后，脂多糖LPS诱导巨噬细胞表型偏移，用RT-PCR法检测巨噬细胞表型标志物的相对表达量；4CCK-8法测定稳定细胞株的增殖活性；5流式细胞术检测巨噬细胞的细胞周期及细胞凋亡；结果：1成功建立稳定干扰KLF4的细胞株，RT-PCR法和Western-blot法鉴定干扰效率达70%以上，；2RT-PCR法结果显示，LPS诱导后，抑制巨噬细胞KLF4的表达使M1型巨噬细胞标志物TNF-α，MCP-1及CCL5的表达降低（P<0.05）；3CCK-8法结果显示，降低巨噬细胞KLF4的表达使细胞增殖活性下降（P<0.05）；4流式细胞术结果显示，抑制巨噬细胞KLF4的表达促进巨噬细胞的凋亡（P<0.05）；结论：1KLF4在实验性肺纤维化发生发展中呈现明显的动态变化；2阿托伐他汀干预博莱霉素致肺纤维化小鼠后KLF4表达明显下调；3KLF4低表达对巨噬细胞的细胞周期无明显影响，但促进其凋亡，抑制其增殖，同时抑制巨噬细胞向M1型偏移；4阿托伐他汀可能通过抑制KLF4的表达来实现其在实验性肺纤维化中的抗炎和抗纤维化作用。

    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样因子4在实验性肺纤维化中的表达及干预研究

摘要5-9ABSTRACT9-13引言14-17    参考文献15-17第一部分 KLF4 在实验性肺纤维化小鼠肺组织中的动态表达17-31    前言17    材料与方法17-23    结果23-24    附图24-27    附表27-28    讨论28-29    小结29    参考文献29-31第二部分 阿托伐他汀对实验性肺纤维化小鼠肺组织中 KLF4 表达的影响31-44    前言31    材料与方法31-34    结果34-36    附图36-40    附表40-41    讨论41-42    小结42    参考文献42-44第三部分 干扰 KLF4 对 RAW264.7 巨噬细胞细胞增殖、凋亡及表型偏移的影响44-57    前言44    材料与方法44-49    结果49-51    附图51-54    附表54-55    讨论55-56    小结56    参考文献56-57结论57-58综述 Krüppel 样因子 4 在肺纤维化中的作用58-65    参考文献61-65致谢65-66个人简历66

  本文关键词：Krüppel样因子4在实验性肺纤维化中的表达及干预研究，由笔耕文化传播整理发布。