本文選題：沙利度胺　切入點(diǎn)：JNK信號通路　出處：《山西醫(yī)科大學(xué)》2013年碩士論文

【摘要】：研究背景：肺纖維化是一類累及肺間質(zhì)、肺泡和（或）細(xì)支氣管的漸進(jìn)性加重的致命性肺部彌漫性疾病。其發(fā)病機(jī)制不清，起病隱匿，病死率高，預(yù)后較差，目前仍缺乏有效的臨床治療手段。所以近年來其發(fā)病機(jī)制和治療成為研究的熱點(diǎn)�，F(xiàn)有研究證實(shí)，肺纖維化的主要病理特征為細(xì)胞外基質(zhì)（extracellularmatrix，ECM）的過度沉積。絲裂原活化蛋白激酶（MAPK）是一種絲／蘇氨酸蛋白激酶，廣泛存在于哺乳動(dòng)物細(xì)胞內(nèi)。c-jun氨基末端激酶（c-junN-terminalkinase，JNK）是重要的MAPK家族成員。有研究表明JNK信號通路在轉(zhuǎn)化生長因子β1（transforminggrowthfactor-β1，TGF-β1）誘導(dǎo)ECM，如纖維連接蛋白、I、III型膠原蛋白等過度聚集的過程中起重要作用。亦有研究顯示，沙利度胺可能通過抑制TGF-β1介導(dǎo)的信號通路來減輕纖BLM誘導(dǎo)的大鼠肺纖維化。但是沙利度胺是否通過下調(diào)JNK信號通路抑制ECM過度沉積，從而減輕BLM誘導(dǎo)的大鼠肺纖維化未見報(bào)道。本實(shí)驗(yàn)我們以Wistar大鼠為研究對象，觀察沙利度胺對肺間質(zhì)纖維化大鼠JNK信號通路及I型膠原蛋白的的影響，從而探討沙利度胺抑制肺間質(zhì)纖維化形成的信號通路機(jī)制，為肺纖維化提供新的臨床治療思路。 目的：以博萊霉素誘導(dǎo)大鼠肺纖維化，建立肺纖維化動(dòng)物模型，通過觀察正常對照組、模型組、沙利度胺組、SP600125組和沙利度胺+SP600125組大鼠肺組織病理學(xué)的改變，檢測大鼠肺組織羥脯氨酸含量，同時(shí)觀察肺組織中I型膠原蛋白和p-JNK蛋白的表達(dá)，分析沙利度胺是否通過抑制JNK信號通路抑制I型膠原蛋白在肺纖維化大鼠肺組織中的過度表達(dá)，從而減輕BLM誘導(dǎo)的大鼠肺間質(zhì)纖維化。 方法：90只大鼠按照隨機(jī)數(shù)字法分為健康對照組(N組)、模型組（M組）、沙利度胺組（T組）、SP600125組（SP組）和沙利度胺+SP600125組（T+SP組），每組18只。M組于氣管內(nèi)滴注BLM0.9%氯化鈉注射液0.3ml（按5mg/kg），造模當(dāng)日腹腔內(nèi)注射DMSO液0.3ml，自造模當(dāng)日起每日給予2ml生理鹽水灌胃；T組、SP組和T+SP組同樣方法造模，T組自造模當(dāng)日起每日給予沙利度胺片灌胃（按100mg/kg，溶于2ml生理鹽水），腹腔內(nèi)注射DMSO液0.3ml；SP組造模當(dāng)日腹腔內(nèi)注射SP600125溶液0.3ml（按15mg/kg，用DMSO溶解至0.3ml），一次性給藥，自造模當(dāng)日起每日給予2ml生理鹽水灌胃；T+SP組造模當(dāng)日腹腔內(nèi)注射SP600125溶液0.3ml（按15mg/kg，用DMSO溶解至0.3ml），并于造模當(dāng)日起每日給予沙利度胺片灌胃（按100mg/kg，溶于2ml生理鹽水）；N組氣管內(nèi)滴注生理鹽水0.3ml，造模當(dāng)日腹腔內(nèi)注射DMSO溶液0.3ml，每日給予2ml生理鹽水灌胃。于造模后第7、14、28天采用腹腔放血法分別處死每組大鼠各6只，打開胸腔，剪取右肺上葉保存于-20℃冰箱，待測羥脯氨酸（Hyp）含量；取右肺下葉，中性甲醛固定，石蠟包埋、切片供病理學(xué)檢測。留取部分肺組織凍存與液氮中，進(jìn)行Western印跡法檢測肺組織中p-JNK及I型膠原蛋白表達(dá)水平。 結(jié)果：1、M組第7天形成典型的肺泡炎改變，第14天炎癥改變減輕，肺間質(zhì)中可見成纖維細(xì)胞增生，，于第28天形成顯著肺纖維化，T組、SP組和T+SP組各時(shí)間點(diǎn)炎癥反應(yīng)及纖維化程度均較M組減輕。2、M組、T組、SP組和T+SP組的Hyp含量隨時(shí)間延長而升高，第28天Hyp含量達(dá)到最高。第14、28天M組、T組、SP組和T+SP組Hyp含量均顯著高于N組（P＜0.01)；T組、SP組和T+SP組Hyp含量較M組顯著減少（P＜0.05)。3、M組各時(shí)間點(diǎn)I型膠原蛋白表達(dá)明顯增多，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05)；T組、SP組和T+SP組I型膠原蛋白表達(dá)顯著減少，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05)；T+SP組I型膠原蛋白表達(dá)均較SP組顯著，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05)。4、M組各時(shí)間點(diǎn)p-JNK蛋白表達(dá)較N組顯著增多，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05)變化趨勢為：第7天開始升高，第14天達(dá)到高峰，第28天開始下降。；T組、SP組和T+SP組p-JNK蛋白表達(dá)較M組顯著減少（P＜0.05)。SP組和T+SP組p-JNK蛋白表達(dá)較T組顯著少，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05)。SP組和T+SP組p-JNK蛋白表達(dá)無顯著差異。 結(jié)論：沙利度胺可能通過抑制JNK信號通路抑制肺纖維化大鼠I型膠原蛋白的過度表達(dá)，從而減輕大鼠肺間質(zhì)纖維化。
[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.
緇撴灉錛

本文編號：1725116