依鲁替尼促进博来霉素介导的肺纤维化作用的机制研究
发布时间：2018-09-13 12:46
【摘要】：目的特发性肺纤维化是一种(IPF)慢性进行性纤维化性间质性肺炎,尽管IPF发病机制迄今未明,但基础和临床研究均提示T细胞、B细胞以及巨噬细胞等免疫细胞过度活化和应答参与IPF的发病(尤其在急性加重期)。依鲁替尼是一种小分子靶向制剂,可选择性地抑制布鲁顿酪氨酸激酶(BTK),对免疫细胞的功能发挥重要的调控作用,临床上多用来治疗淋巴细胞白血病,近来也有用于治疗免疫相关疾病如免疫排斥的报道。然而,依鲁替尼是否能够通过免疫抑制的机制延缓IPF进程还未有研究。本课题旨在研究依鲁替尼在博莱霉素诱导的小鼠肺纤维化模型中的干预作用,并探讨相关机制。材料和方法32只C57BL/6J小鼠分为实验组(n=16)和对照组(n=16),两组小鼠均经气管滴入博莱霉素(3mg/kg)建立小鼠肺纤维化模型,实验第一天(d1)起实验组小鼠隔天给予10mg/kg依鲁替尼口饲,对照组相同条件下给予等量的生理盐水。d14处死小鼠,结扎左肺上叶,取出肺组织行HE染色和Masson染色,免疫组化分析α-SMA以及肺组织羟脯氨酸法评估纤维化程度。余肺进行肺泡灌洗,流式分析、EILSA、RT-PCR以及Western blot检测肺泡灌洗液(BALF)及肺组织中细胞组分细胞组成、蛋白浓度(Fn、Coll、Vinmentin、Occludin、E-cadherin 及 Snail)、细胞因子(TGF-β、TNF、IL-6、IL-17)以及相关信号通路的表达(TGF-β/Smad)。结果与对照组比较,实验组模型小鼠肺泡炎症和肺纤维化程度明显加重,肺组织中α-SMA的表达水平及BALF中蛋白浓度明显增高,肺组织中TGF-β的mRNA和蛋白表达升高,肺上皮细胞中Col1、Fn、Vimentin及Snail表达量上升,TGF-β下游Smad磷酸化水平增高。结论依鲁替尼可能通过增强肺组织炎症应答,调控TGF-β/Smad信号通路促进EMT以及诱导肌成纤维细胞增殖分化等机制,加速IPF模型小鼠的肺纤维化进程。
[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 伪 -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 伪 -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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563
，
本文编号：2241214
[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 伪 -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 伪 -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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563
，
本文编号：2241214
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