Notch信号与BLM诱导大鼠肺纤维化

发布时间：2018-01-16 06:14

  本文关键词：Notch信号与BLM诱导大鼠肺纤维化　出处：《第四军医大学》2012年博士论文　论文类型：学位论文

  更多相关文章： 博莱霉素 肺纤维化 肺微血管内皮细胞 成纤维细胞 肌成纤维细胞 Notch信号 结缔组织生长因子 血管内皮生长因子

【摘要】：背景 肺微血管内皮细胞（Pulmonary microvascular endothelial cells，PMVECs）是肺气血屏障的重要组成结构，生理情况下维持人体正常的呼吸交换功能，其功能变化对维持血管正常结构以及肺组织损伤后的修复起重要作用。在肺纤维化（Pulmonary fibrosis，PF）的形成过程中，成纤维细胞（Fibroblast，Fb）的激活及转化为肌成纤维细胞（Myofibroblast，MFb）是肺间质内基质成分过度沉积的关键因素。已经证实，肺内MFb有四种来源：间质内的Fb在炎细胞分泌细胞因子作用下转化成MFb；骨髓干细胞转化为MFb；肺泡上皮细胞间充质转分化（Epithelial-mesenchymal transition，EMT）；血管内皮细胞间充质转分化（Endothelial-mesenchymal transition，EndoMT）。其中EndoMT在肺纤维化形成中的地位和作用已经引起广泛关注。 既往的研究中发现，PF患者及实验动物模型肺组织血管密度分布不均，在纤维化区周边存在异常的新生血管，而中心区血管退化[1]。增生的PMVECs分化不全，形成的微血管血液灌注不良，不能发挥正常的生理功能。我们前期实验发现，博莱霉素（Bleomycin，BLM）致PF大鼠PMVECs增殖活跃并分泌促纤维化的细胞因子，如转化生长因子-β1（Transforminggrowth factor，TGF-β1）和结缔组织生长因子（Connective tissue growthfactor，CTGF），而且与PMVECs共培养的Fbs可显著增加平滑肌肌动蛋白（smooth muscle actin，a-SMA）的表达[2]。目前，PMVECs的这种异常增殖、分泌致纤维化因子及EndoMT的调控因素不明。 Notch信号通路广泛参与机体发育、细胞分化及稳态调节。近年来发现，Notch对EC的增殖和分化具有重要的调节作用。Notch激活可通过抑制血管内皮生长因子受体2（Vascular endothelial growth factor receptor2，VEGFR2/Flk-1/KDR）的表达特异性调控由VEGF诱导的EC增殖与分化，使血管正常发育并维护其生理功能。 综上所述，MFb是肺纤维化形成的标志性细胞，而MFb的来源，，包括PMVECs来源的EndoMT，具有多样化的特点。基于Notch信号在调控增殖的微血管中的作用，我们假设BLM可能降低或损伤了多种细胞的Notch信号通路，致使其成为调控增殖状态的PMVECs及Fbs转化为EndoMT及MFb的前提和基础。 方法和目的 本实验采用气管内注入BLM诱导SD大鼠PF模型，气管内注入等体积生理盐水作为对照。分别于给药后第7天和第14天处死大鼠，取外周肺组织原代培养PMVECs和Fbs并鉴定，余肺组织常规固定、包埋、切片、VG染色。采用免疫组织化学法检测肺组织中Hes1、KDR的表达；细胞免疫荧光化学法检测PMVECs中PCNA的表达水平；蛋白印记（western blot）、实时定量PCR（Real-time PCR）检测各组PMVECs中Notch信号通路重要分子Hes1、Notch1、Jag1、Dll4的蛋白及mRNA水平的表达；Western Blot检测Fbs中Notch信号相关分子的蛋白表达水平；结合各组PCNA和-SMA的表达情况，以明确PF时Notch信号通路发生的变化，以及对PMVECs和Fbs增殖、分化的影响。 结果 1.细胞免疫荧光及Western Blot检测BLM组PMVECs的PCNA蛋白表达上调且α-SMA表达增加，免疫组织荧光显示BLM组肺内有共表达α-SMA和VWF的细胞存在，证实BLM大鼠PMVECs不仅处于增殖状态，而且表型已发生转变。 2.免疫组织化学、Western Blot、Real-time PCR检测显示BLM组PMVECs的Hes1表达下调，KDR表达上调，Jag1表达明显上调，而Dll4表达较正常组低，提示肺纤维化时PMVECs的Notch信号通路抑制且配体表达异常。 3. Western Blot检测显示BLM组Fbs的Hes1表达下调，Jag1上调，同时PCNA及-SMA的表达水平增高，提示肺纤维化时Notch信号的抑制可能与MFb的产生有关。 结论 1. PMVECs的EndoMT及Fbs向MFb转分化可能是BLM大鼠肺纤维化形成的重要原因之一，而Notch信号通路抑制是BLM大鼠异常增殖的PMVECs及Fbs转分化为EndoMT及MFb的基础。 2. Notch配体Jag1高表达和Dll4低表达可能造成VEGF-Notch-KDR负反馈调节途径异常。 3. Jag1的高表达可能与Fbs的增殖及转分化有关。
[Abstract]:background
Pulmonary microvascular endothelial cells (Pulmonary microvascular endothelial cells, PMVECs) is an important component of the pulmonary blood barrier, physiological conditions to maintain normal human respiratory exchange function, its function changes to maintain the normal structure of blood vessels and repair of lung tissue injury plays an important role in pulmonary fibrosis (Pulmonary fibrosis, PF) forming process in fibroblasts (Fibroblast, Fb) activated and transformed into myofibroblasts (Myofibroblast, MFb) is a key factor in lung interstitial matrix. The excessive deposition of MFb in the lung has confirmed that there are four sources of interstitial Fb cytokine secretion function in inflammatory cells converted under MFb; bone marrow stem cells into MFb cells; alveolar epithelial mesenchymal transition (Epithelial-mesenchymal transition, EMT); vascular endothelial mesenchymal transition (Endothelial-mesenchymal transition, E NdoMT). The role and role of EndoMT in the formation of pulmonary fibrosis has attracted wide attention.
Found in previous studies, lung tissue of patients and experimental animal models of PF vascular density distribution is uneven, there is abnormal neovascularization in fibrotic areas surrounding the differentiation of PMVECs, and the central area of vascular proliferation of [1]. degradation is not complete, the formation of the microvascular blood perfusion, can not play a normal physiological function. We found that pre experiment. Bleomycin (Bleomycin, BLM) PMVECs PF induced proliferation of rat and active cytokine secretion of promoting fibrosis, such as transforming growth factor beta 1 (Transforminggrowth factor, TGF- beta 1) and connective tissue growth factor (Connective tissue, growthfactor, CTGF) and PMVECs co cultured with Fbs significantly increased smooth muscle actin (smooth muscle actin, a-SMA) the expression of [2]. at present, the abnormal proliferation of PMVECs, secretion fibrogenetic factors and EndoMT factors is unknown.
Notch signaling pathway is widely involved in organism development, cell differentiation and homeostasis. In recent years, Notch on EC proliferation and differentiation plays an important role in the regulation of.Notch activation through inhibition of vascular endothelial growth factor receptor 2 (Vascular endothelial growth factor receptor2, VEGFR2/Flk-1/KDR) expression of specific regulation by VEGF induced EC proliferation and differentiation. The normal development of vascular and maintain its physiological function.
In summary, MFb is the hallmark of cell lung fibrosis, and MFb source, including the PMVECs source EndoMT, has diversified characteristics. The role of Notch signal in the regulation of microvascular proliferation in based on, we hypothesized that BLM may reduce or damage the Notch signal pathway in different cells, which become PMVECs and Fbs regulation the proliferation state of transformation is the premise and basis of EndoMT and MFb.
Methods and purposes
This experiment by intratracheal instillation of SD rat PF model induced by BLM, intratracheal injection of saline as control. The medicine was given after seventh days and fourteenth days, the rats were sacrificed and the peripheral lung tissue cultured in PMVECs and Fbs and identified more than lung tissue were fixed, embedded, sliced, VG Hes1 staining. The lung tissues were detected by immunohistochemical method, the expression of KDR; the expression of PCNA was detected in PMVECs cells by immunofluorescence method; Western blot (Western blot), real time quantitative PCR (Real-time PCR) detection of Notch signal pathway in PMVECs were important molecules Hes1, Notch1, Jag1, mRNA and protein expression level Dll4; expression of Notch related signaling molecules Western Blot detection in Fbs binding protein; expression of PCNA was -SMA and the changes in the Notch signal pathway occur explicitly at PF, and the PMVECs and Fbs proliferation in vitro.
Result
Expression of 1. cell immunofluorescence and Western Blot detection of BLM group PMVECs upregulation of PCNA protein alpha and increase the expression of -SMA, immunohistochemistry showed that BLM group lung has co expressed -SMA and VWF alpha cells confirmed that BLM rats PMVECs not only in proliferation, phenotype and has changed.
2. immunohistochemistry, Western Blot, Real-time PCR detection showed that Hes1 expression in PMVECs group was down regulated, KDR expression was up-regulated, Jag1 expression was upregulated, while Dll4 expression was lower than that in normal group, indicating that PMVECs signaling pathway was inhibited and ligand expression was abnormal in pulmonary fibrosis.
3. Western Blot detection showed that the expression of Fbs in BLM group was down regulated, Jag1 was upregulated, and the expression level of PCNA and -SMA increased, suggesting that inhibition of Notch signal in pulmonary fibrosis may be related to the production of MFb.
conclusion
1. PMVECs EndoMT and Fbs transdifferentiation to MFb may be one of the important reasons for the formation of pulmonary fibrosis in BLM rats. Notch signal pathway inhibition is the basis for PMVECs proliferation and Fbs transformation into BLM and EndoMT in BLM rats.
The high expression of the 2. Notch ligand Jag1 and the low expression of Dll4 may result in the abnormal regulation of VEGF-Notch-KDR negative feedback.
The high expression of 3. Jag1 may be related to the proliferation and transdifferentiation of Fbs.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【共引文献】

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1 邓维叶;高云飞;陈艳峰;李浩;杨远忠;郭朱明;;Notch-1在木村病中的表达及意义[J];中华临床医师杂志(电子版);2014年05期

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1 何飞;Notch信号对小鼠肝纤维化作用的研究[D];第四军医大学;2011年

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相关硕士学位论文 前1条

1 张建平;DLL1基因在小鼠B16黑色素瘤形成中的作用研究[D];第四军医大学;2011年

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