沙利度胺通过抑制JNK信号通路抑制肺纤维化大鼠I型胶原蛋白的过度表达

发布时间：2018-04-09 05:37

  本文选题：沙利度胺　切入点：JNK信号通路　出处：《山西医科大学》2013年硕士论文

【摘要】：研究背景：肺纤维化是一类累及肺间质、肺泡和（或）细支气管的渐进性加重的致命性肺部弥漫性疾病。其发病机制不清，起病隐匿，病死率高，预后较差，目前仍缺乏有效的临床治疗手段。所以近年来其发病机制和治疗成为研究的热点。现有研究证实，肺纤维化的主要病理特征为细胞外基质（extracellularmatrix，ECM）的过度沉积。丝裂原活化蛋白激酶（MAPK）是一种丝／苏氨酸蛋白激酶，广泛存在于哺乳动物细胞内。c-jun氨基末端激酶（c-junN-terminalkinase，JNK）是重要的MAPK家族成员。有研究表明JNK信号通路在转化生长因子β1（transforminggrowthfactor-β1，TGF-β1）诱导ECM，如纤维连接蛋白、I、III型胶原蛋白等过度聚集的过程中起重要作用。亦有研究显示，沙利度胺可能通过抑制TGF-β1介导的信号通路来减轻纤BLM诱导的大鼠肺纤维化。但是沙利度胺是否通过下调JNK信号通路抑制ECM过度沉积，从而减轻BLM诱导的大鼠肺纤维化未见报道。本实验我们以Wistar大鼠为研究对象，观察沙利度胺对肺间质纤维化大鼠JNK信号通路及I型胶原蛋白的的影响，从而探讨沙利度胺抑制肺间质纤维化形成的信号通路机制，为肺纤维化提供新的临床治疗思路。 目的：以博莱霉素诱导大鼠肺纤维化，建立肺纤维化动物模型，通过观察正常对照组、模型组、沙利度胺组、SP600125组和沙利度胺+SP600125组大鼠肺组织病理学的改变，检测大鼠肺组织羟脯氨酸含量，同时观察肺组织中I型胶原蛋白和p-JNK蛋白的表达，分析沙利度胺是否通过抑制JNK信号通路抑制I型胶原蛋白在肺纤维化大鼠肺组织中的过度表达，从而减轻BLM诱导的大鼠肺间质纤维化。 方法：90只大鼠按照随机数字法分为健康对照组(N组)、模型组（M组）、沙利度胺组（T组）、SP600125组（SP组）和沙利度胺+SP600125组（T+SP组），每组18只。M组于气管内滴注BLM0.9%氯化钠注射液0.3ml（按5mg/kg），造模当日腹腔内注射DMSO液0.3ml，自造模当日起每日给予2ml生理盐水灌胃；T组、SP组和T+SP组同样方法造模，T组自造模当日起每日给予沙利度胺片灌胃（按100mg/kg，溶于2ml生理盐水），腹腔内注射DMSO液0.3ml；SP组造模当日腹腔内注射SP600125溶液0.3ml（按15mg/kg，用DMSO溶解至0.3ml），一次性给药，自造模当日起每日给予2ml生理盐水灌胃；T+SP组造模当日腹腔内注射SP600125溶液0.3ml（按15mg/kg，用DMSO溶解至0.3ml），并于造模当日起每日给予沙利度胺片灌胃（按100mg/kg，溶于2ml生理盐水）；N组气管内滴注生理盐水0.3ml，造模当日腹腔内注射DMSO溶液0.3ml，每日给予2ml生理盐水灌胃。于造模后第7、14、28天采用腹腔放血法分别处死每组大鼠各6只，打开胸腔，剪取右肺上叶保存于-20℃冰箱，待测羟脯氨酸（Hyp）含量；取右肺下叶，中性甲醛固定，石蜡包埋、切片供病理学检测。留取部分肺组织冻存与液氮中，进行Western印迹法检测肺组织中p-JNK及I型胶原蛋白表达水平。 结果：1、M组第7天形成典型的肺泡炎改变，第14天炎症改变减轻，肺间质中可见成纤维细胞增生，，于第28天形成显著肺纤维化，T组、SP组和T+SP组各时间点炎症反应及纤维化程度均较M组减轻。2、M组、T组、SP组和T+SP组的Hyp含量随时间延长而升高，第28天Hyp含量达到最高。第14、28天M组、T组、SP组和T+SP组Hyp含量均显著高于N组（P＜0.01)；T组、SP组和T+SP组Hyp含量较M组显著减少（P＜0.05)。3、M组各时间点I型胶原蛋白表达明显增多，差异有统计学意义（P＜0.05)；T组、SP组和T+SP组I型胶原蛋白表达显著减少，差异有统计学意义（P＜0.05)；T+SP组I型胶原蛋白表达均较SP组显著，差异有统计学意义（P＜0.05)。4、M组各时间点p-JNK蛋白表达较N组显著增多，差异有统计学意义（P＜0.05)变化趋势为：第7天开始升高，第14天达到高峰，第28天开始下降。；T组、SP组和T+SP组p-JNK蛋白表达较M组显著减少（P＜0.05)。SP组和T+SP组p-JNK蛋白表达较T组显著少，差异有统计学意义（P＜0.05)。SP组和T+SP组p-JNK蛋白表达无显著差异。 结论：沙利度胺可能通过抑制JNK信号通路抑制肺纤维化大鼠I型胶原蛋白的过度表达，从而减轻大鼠肺间质纤维化。
[Abstract]:Background: pulmonary fibrosis is a kind of interstitial lung involvement, alveolar and bronchiolar (or) a progressive worsening of fatal diffuse lung disease. Its pathogenesis is not clear, onset and high mortality rate, poor prognosis, there is still lack of effective treatment. So in recent years and its pathogenesis the treatment has become the focus of research. The existing research confirmed that the main pathological features of pulmonary fibrosis to extracellular matrix (extracellularmatrix, ECM). The excessive deposition of mitogen activated protein kinase (MAPK) is a serine / threonine protein kinase, widely exist in mammalian cells.C-jun N-terminal kinase (c-junN-terminalkinase, JNK) is important a member of the MAPK family. Studies have shown that the JNK signaling pathway in transforming growth factor beta 1 (transforminggrowthfactor- beta 1, beta 1 TGF-) induced by ECM, such as fibronectin, I, type III collagen excessive poly Play an important role in the process of collecting. Also studies have shown that thalidomide may reduce fiber BLM induced pulmonary fibrosis in rats by inhibiting signal transduction mediated by TGF- beta 1. But whether thalidomide by inhibiting the excessive deposition of ECM down-regulation of JNK signaling pathway, thereby reducing the BLM induced pulmonary fibrosis in rats was reported. In this experiment, we the Wistar rats as the research object, to observe the effect of thalidomide on pulmonary fibrosis in rats JNK signal pathway and type I collagen, so as to explore the signal transduction mechanism of thalidomide inhibits pulmonary interstitial fibrosis, and provide new ideas for clinical treatment of pulmonary fibrosis.
Objective: pulmonary fibrosis rats induced by bleomycin, establish the animal model of pulmonary fibrosis, through the observation of the normal control group, model group, thalidomide group, pathology group and thalidomide group +SP600125 rats lung tissue SP600125 change detection of rat lung tissue hydroxyproline content at the same time, to observe the expression of I in lung tissue, collagen and p-JNK protein the analysis of whether thalidomide through excessive inhibition of expression of type I collagen in lung tissue of rats with pulmonary fibrosis in the inhibition of the JNK signaling pathway, thereby reducing the BLM induced pulmonary fibrosis in rats.
Methods: 90 rats were randomly divided into normal control group (N group), model group (group M), thalidomide group (T group), SP600125 group (group SP) and thalidomide group +SP600125 (T+SP group), 18 rats in each group.M in intratracheal instillation of BLM0.9% (according to Sodium Chloride Injection 0.3ml 5mg/kg), was injected i.p with DMSO liquid 0.3ml, since the date of the modeling daily given 2ml saline; T group, SP group and T+SP group the same modeling method, T group since the date of the modeling daily give Thalidomide Tablets gavage (according to the 100mg/kg, dissolved in 2ml saline), injection of DMSO 0.3ml intraperitoneal; group SP was injected i.p SP600125 solution 0.3ml (15mg/kg, dissolved in DMSO to 0.3ml), a one-time delivery, since the date of the modeling daily given 2ml saline; group T+SP was injected i.p SP600125 solution 0.3ml (15mg/kg, DMSO, and dissolved to 0.3ml) modeling The date given daily gavage (Thalidomide Tablets press 100mg/kg, dissolved in 2ml saline); N group intratracheal instillation of normal saline 0.3ml was injected i.p with DMSO 0.3ml solution, a daily dose of 2ml saline. In celiac bloodletting method using the 7,14,28 day after modeling respectively sacrificed all rats in each group 6, open the chest, clipping the upper lobe of the right lung preservation in -20 C refrigerator, for determination of hydroxyproline (Hyp) content; the lower lobe of right lung, neutral formalin fixed, paraffin embedded, sliced for pathological examination. Take a part of lung tissue cryopreservation with liquid nitrogen, the expression level of p-JNK was detected in the lung tissue of Western Western blotting and type I collagen.
缁撴灉锛

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