本文關(guān)鍵詞：Notch信號與BLM誘導(dǎo)大鼠肺纖維化　出處：《第四軍醫(yī)大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 博萊霉素 肺纖維化 肺微血管內(nèi)皮細胞 成纖維細胞 肌成纖維細胞 Notch信號 結(jié)締組織生長因子 血管內(nèi)皮生長因子

【摘要】：背景 肺微血管內(nèi)皮細胞（Pulmonary microvascular endothelial cells，PMVECs）是肺氣血屏障的重要組成結(jié)構(gòu)，生理情況下維持人體正常的呼吸交換功能，其功能變化對維持血管正常結(jié)構(gòu)以及肺組織損傷后的修復(fù)起重要作用。在肺纖維化（Pulmonary fibrosis，PF）的形成過程中，成纖維細胞（Fibroblast，F(xiàn)b）的激活及轉(zhuǎn)化為肌成纖維細胞（Myofibroblast，MFb）是肺間質(zhì)內(nèi)基質(zhì)成分過度沉積的關(guān)鍵因素。已經(jīng)證實，肺內(nèi)MFb有四種來源：間質(zhì)內(nèi)的Fb在炎細胞分泌細胞因子作用下轉(zhuǎn)化成MFb；骨髓干細胞轉(zhuǎn)化為MFb；肺泡上皮細胞間充質(zhì)轉(zhuǎn)分化（Epithelial-mesenchymal transition，EMT）；血管內(nèi)皮細胞間充質(zhì)轉(zhuǎn)分化（Endothelial-mesenchymal transition，EndoMT）。其中EndoMT在肺纖維化形成中的地位和作用已經(jīng)引起廣泛關(guān)注。 既往的研究中發(fā)現(xiàn)，PF患者及實驗動物模型肺組織血管密度分布不均，在纖維化區(qū)周邊存在異常的新生血管，而中心區(qū)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，不能發(fā)揮正常的生理功能。我們前期實驗發(fā)現(xiàn)，博萊霉素（Bleomycin，BLM）致PF大鼠PMVECs增殖活躍并分泌促纖維化的細胞因子，如轉(zhuǎn)化生長因子-β1（Transforminggrowth factor，TGF-β1）和結(jié)締組織生長因子（Connective tissue growthfactor，CTGF），而且與PMVECs共培養(yǎng)的Fbs可顯著增加平滑肌肌動蛋白（smooth muscle actin，a-SMA）的表達[2]。目前，PMVECs的這種異常增殖、分泌致纖維化因子及EndoMT的調(diào)控因素不明。 Notch信號通路廣泛參與機體發(fā)育、細胞分化及穩(wěn)態(tài)調(diào)節(jié)。近年來發(fā)現(xiàn)，Notch對EC的增殖和分化具有重要的調(diào)節(jié)作用。Notch激活可通過抑制血管內(nèi)皮生長因子受體2（Vascular endothelial growth factor receptor2，VEGFR2/Flk-1/KDR）的表達特異性調(diào)控由VEGF誘導(dǎo)的EC增殖與分化，使血管正常發(fā)育并維護其生理功能。 綜上所述，MFb是肺纖維化形成的標志性細胞，而MFb的來源，，包括PMVECs來源的EndoMT，具有多樣化的特點�；贜otch信號在調(diào)控增殖的微血管中的作用，我們假設(shè)BLM可能降低或損傷了多種細胞的Notch信號通路，致使其成為調(diào)控增殖狀態(tài)的PMVECs及Fbs轉(zhuǎn)化為EndoMT及MFb的前提和基礎(chǔ)。 方法和目的 本實驗采用氣管內(nèi)注入BLM誘導(dǎo)SD大鼠PF模型，氣管內(nèi)注入等體積生理鹽水作為對照。分別于給藥后第7天和第14天處死大鼠，取外周肺組織原代培養(yǎng)PMVECs和Fbs并鑒定，余肺組織常規(guī)固定、包埋、切片、VG染色。采用免疫組織化學(xué)法檢測肺組織中Hes1、KDR的表達；細胞免疫熒光化學(xué)法檢測PMVECs中PCNA的表達水平；蛋白印記（western blot）、實時定量PCR（Real-time PCR）檢測各組PMVECs中Notch信號通路重要分子Hes1、Notch1、Jag1、Dll4的蛋白及mRNA水平的表達；Western Blot檢測Fbs中Notch信號相關(guān)分子的蛋白表達水平；結(jié)合各組PCNA和-SMA的表達情況，以明確PF時Notch信號通路發(fā)生的變化，以及對PMVECs和Fbs增殖、分化的影響。 結(jié)果 1.細胞免疫熒光及Western Blot檢測BLM組PMVECs的PCNA蛋白表達上調(diào)且α-SMA表達增加，免疫組織熒光顯示BLM組肺內(nèi)有共表達α-SMA和VWF的細胞存在，證實BLM大鼠PMVECs不僅處于增殖狀態(tài)，而且表型已發(fā)生轉(zhuǎn)變。 2.免疫組織化學(xué)、Western Blot、Real-time PCR檢測顯示BLM組PMVECs的Hes1表達下調(diào)，KDR表達上調(diào)，Jag1表達明顯上調(diào)，而Dll4表達較正常組低，提示肺纖維化時PMVECs的Notch信號通路抑制且配體表達異常。 3. Western Blot檢測顯示BLM組Fbs的Hes1表達下調(diào)，Jag1上調(diào)，同時PCNA及-SMA的表達水平增高，提示肺纖維化時Notch信號的抑制可能與MFb的產(chǎn)生有關(guān)。 結(jié)論 1. PMVECs的EndoMT及Fbs向MFb轉(zhuǎn)分化可能是BLM大鼠肺纖維化形成的重要原因之一，而Notch信號通路抑制是BLM大鼠異常增殖的PMVECs及Fbs轉(zhuǎn)分化為EndoMT及MFb的基礎(chǔ)。 2. Notch配體Jag1高表達和Dll4低表達可能造成VEGF-Notch-KDR負反饋調(diào)節(jié)途徑異常。 3. Jag1的高表達可能與Fbs的增殖及轉(zhuǎn)分化有關(guān)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R363

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本文編號：1431883