【摘要】：目的特發(fā)性肺纖維化是一種(IPF)慢性進(jìn)行性纖維化性間質(zhì)性肺炎,盡管IPF發(fā)病機(jī)制迄今未明,但基礎(chǔ)和臨床研究均提示T細(xì)胞、B細(xì)胞以及巨噬細(xì)胞等免疫細(xì)胞過(guò)度活化和應(yīng)答參與IPF的發(fā)病(尤其在急性加重期)。依魯替尼是一種小分子靶向制劑,可選擇性地抑制布魯頓酪氨酸激酶(BTK),對(duì)免疫細(xì)胞的功能發(fā)揮重要的調(diào)控作用,臨床上多用來(lái)治療淋巴細(xì)胞白血病,近來(lái)也有用于治療免疫相關(guān)疾病如免疫排斥的報(bào)道。然而,依魯替尼是否能夠通過(guò)免疫抑制的機(jī)制延緩IPF進(jìn)程還未有研究。本課題旨在研究依魯替尼在博萊霉素誘導(dǎo)的小鼠肺纖維化模型中的干預(yù)作用,并探討相關(guān)機(jī)制。材料和方法32只C57BL/6J小鼠分為實(shí)驗(yàn)組(n=16)和對(duì)照組(n=16),兩組小鼠均經(jīng)氣管滴入博萊霉素(3mg/kg)建立小鼠肺纖維化模型,實(shí)驗(yàn)第一天(d1)起實(shí)驗(yàn)組小鼠隔天給予10mg/kg依魯替尼口飼,對(duì)照組相同條件下給予等量的生理鹽水。d14處死小鼠,結(jié)扎左肺上葉,取出肺組織行HE染色和Masson染色,免疫組化分析α-SMA以及肺組織羥脯氨酸法評(píng)估纖維化程度。余肺進(jìn)行肺泡灌洗,流式分析、EILSA、RT-PCR以及Western blot檢測(cè)肺泡灌洗液(BALF)及肺組織中細(xì)胞組分細(xì)胞組成、蛋白濃度(Fn、Coll、Vinmentin、Occludin、E-cadherin 及 Snail)、細(xì)胞因子(TGF-β、TNF、IL-6、IL-17)以及相關(guān)信號(hào)通路的表達(dá)(TGF-β/Smad)。結(jié)果與對(duì)照組比較,實(shí)驗(yàn)組模型小鼠肺泡炎癥和肺纖維化程度明顯加重,肺組織中α-SMA的表達(dá)水平及BALF中蛋白濃度明顯增高,肺組織中TGF-β的mRNA和蛋白表達(dá)升高,肺上皮細(xì)胞中Col1、Fn、Vimentin及Snail表達(dá)量上升,TGF-β下游Smad磷酸化水平增高。結(jié)論依魯替尼可能通過(guò)增強(qiáng)肺組織炎癥應(yīng)答,調(diào)控TGF-β/Smad信號(hào)通路促進(jìn)EMT以及誘導(dǎo)肌成纖維細(xì)胞增殖分化等機(jī)制,加速IPF模型小鼠的肺纖維化進(jìn)程。
[Abstract]:Objective Idiopathic pulmonary fibrosis is a chronic progressive interstitial pneumonia of (IPF), although the pathogenesis of IPF is unknown. But both basic and clinical studies suggest that T cell B cells, macrophages and other immune cells are over-activated and responsive to the pathogenesis of IPF (especially in acute exacerbation). Irutinib is a small molecular targeted preparation that selectively inhibits the role of Bruton tyrosine kinase (BTK), in regulating the function of immune cells and is widely used in the treatment of lymphoblastic leukemia. There have also been recent reports of treatment for immune-related diseases such as immune rejection. However, whether Irutinib can delay the progress of IPF through immunosuppressive mechanisms has not been studied. The purpose of this study was to investigate the effects of ilutinib on bleomycin-induced pulmonary fibrosis in mice and to explore the related mechanisms. Materials and methods Thirty-two C57BL/6J mice were divided into two groups: experimental group (n = 16) and control group (n = 16). Pulmonary fibrosis models were established by intratracheal instillation of bleomycin (3mg/kg) in both groups. Mice in the experimental group were given 10mg/kg erutini orally every other day from the first day of the experiment (day 1). In the control group, the mice were sacrificed with the same amount of normal saline on day 14. The left upper lobe of lung was ligated. The lung tissues were stained with HE and Masson. The degree of fibrosis was evaluated by immunohistochemical analysis of 偽 -SMA and hydroxyproline in lung tissue. The alveolar lavage was performed in the rest of the lungs. Flow cytometry (RT-PCR) and Western blot were used to detect the cellular composition, protein concentration (Fn,Coll,Vinmentin,Occludin,E-cadherin, TGF- 尾 -TNF-IL-6IL-17) and the expression of related signal pathways (TGF- 尾 -Smad) in the alveolar lavage fluid (BALF) and lung tissue. Results compared with the control group, the degree of alveolar inflammation and pulmonary fibrosis in the experimental group was significantly increased, the expression of 偽 -SMA in lung tissue and the protein concentration in BALF were significantly increased, and the mRNA and protein expression of TGF- 尾 in lung tissue were increased. The expression of Col1,Fn,Vimentin and Snail in lung epithelial cells increased and the level of Smad phosphorylation downstream of TGF- 尾 increased. Conclusion Irutinib may accelerate the process of pulmonary fibrosis in IPF model mice by enhancing the inflammatory response of lung tissue, regulating the TGF- 尾 / Smad signaling pathway to promote EMT and induce the proliferation and differentiation of myofibroblasts.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R563
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本文編號(hào)：2241214