JNK與Smad信號通路在小鼠肺纖維化中相互作用的研究及對Ⅰ型膠原表達的影響
論著
作者：秦世鵬第
[摘要] 目的 探討JNK與Smad途徑介導小鼠肺纖維化中Ⅰ型膠原的表達及二者相互作用的研究。 方法 選擇雄性野生健康C57BL/6小鼠96隻，隨機分為正常組（N組）、模型組（M組）、p-Smad3阻斷組（SB組）、JNK阻斷組（SP組），每組24隻。M組、SB組及SP組氣管內滴注博萊黴素3.5 mg/kg誘導肺纖維化模型，於造模後給予相應藥物，N組給予等劑量生理鹽水。每組分別於造模後第7、14、28天隨機處死8隻小鼠，取肺組織病理切片行HE染色和Masson染色，觀察肺泡炎和纖維化程度；堿水解法檢測肺組織羥脯氨酸（Hyp）含量；免疫組化法測定肺組織Ι型膠原、p-JNK及p-Smad蛋白含量，各組進行對比。 結果 M組第7天肺泡炎症明顯，第28天纖維化改變顯著；隨著時間推移，羥脯氨酸含量呈逐漸上升趨勢，第28天含量最高。SB組、SP組與M組比較，肺泡炎症及纖維化程度均有所減輕；第14、28天Hyp含量稍降低，Ⅰ型膠原、p-JNK及p-Smad蛋白的表達有所減少（P
[關鍵詞] 肺纖維化；轉化生長因子β1；JNK信號通路；Smad信號通路；Ⅰ型膠原
[中圖分類號] R563.9 [文獻標識碼] A [文章編號] 1673-9701（2015）09-0001-05
Study of JNK and Smad signal pathways interaction in pulmonary fibrosis in mice and the influence on the expression of type I collagen
QIN Shipeng1 LIU Xuejun2 WANG Xiaohui2 ZHONG Jianke2 DU Yufeng2
1.Shanxi Medical University，Taiyuan 030001，China；2.Department of Geriatrics，the First Hospital of Shanxi Medical University，Taiyuan 030001，China
[Abstract] Objective To investigate the expression of type Ι collagen in JNK and Smad mediated pulmonary fibrosis of mice and study the interactions between the two mediation. Methods Ninety-six healthy wild male C57BL/6 mice were randomLy divided into four groups，the _disibledevent=24）. M group，SB group and SP group were endo-tracheal driped by bleomycin（3.5 mg/kg） to estabish pulmonary fibrosis model and then given corres-ponding drugs， while the N group was given isodose physiological saline. Eight mice in each group were randomly killed on the 7th，14th，28th. The degree of alveolitis and pulmonary fibrosis by the mice lung tissue pathological in HE staining and Masson staining were observed，lung tissue hydroxyproline（Hyp） content by alkaline hydrolysis method were and dected each group’s lung tissue of type I collagen，p-JNK and p-Smad protein content by immunohistochemical determination were compared. Results The degree of pulmonary alveolitis of M group was obvious on the 7th day after modeling and the fibrosis was reduced significantly on the 28th day. The Hyp increased gradually and reached the maximum level on the 28th day. The pulmonary alveolitis and fibrosis of lung tissue in SB，SP and N group were in a relatively low degree，compared with M group. The Hyp ration of these groups were on a slightly lower level；the expression of type I collage，p-JNK and p-Smad protein were reduced（P
[Key words] Pulmonary fibrosis；Transforming growth factor β1；JNK signaling pathway；Smad signaling pathway；Type Ι collagen
肺纖維化（pulmonary fibrosis，PF）是一種早期以肺泡炎症，後期以大量成纖維細胞（fibroblast，FB）異常增殖和細胞外基質（extracellular matrix，ECM）大量沉積為主要表現特點的肺間質疾病[1]。轉化生長因子β1（TGF-β1）是重要的致纖維化因子，通過多個信號通路發揮作用，JNK及Smad信號通路是其下遊的重要信號通路，p-Smad3高表達與炎症纖維化的發展密切相關[2]。Ⅰ型膠原是ECM中反映肺纖維化程度的主要指標，本研究主要使用特異性阻滯劑阻斷Smad及JNK信號通路後，觀察Ⅰ型膠原的變化程度以及兩條通路的相互關聯。
1 材料與方法
1.1 材料來源
選取18～20 g雄性野生C57BL/6小鼠96隻，2014年1月購於中國人民解放軍軍事醫學科學院實驗動物中心，於山西醫科大學動物實驗中心飼養，SPF級。博萊黴素（日本化藥株式會社），SP600125、SB431542及二甲基亞碸（DMSO）溶液（美國sigma公司）；Masson染液試劑盒、羥脯氨酸試劑盒（南京建成生物工程研究所）；兔抗鼠p-JNK、p-Smad3及Ⅰ型膠原多克隆抗體（美國Bioworld公司）；SABC試劑盒、二氨基聯苯胺（DAB）顯色劑（武漢博士德公司）。
1.2 模型分組及處理
選擇雄性野生健康C57BL/6小鼠96隻，隨機分為正常組（N組）、模型組（M組）、p-Smad3阻斷組（SB組）和JNK阻斷組（SP組），每組24隻。N組給予氣管內滴注0.9%氯化鈉注射液0.04 mL，其餘三組氣管內滴注博來黴素0.04 mL（3.5 mg/kg）建立肺纖維化模型。SP組采用SP600125溶於二甲基亞碸（DMSO）溶液，腹腔注射0.03 mL（15 mg/kg），於造模當日注射；其餘各組腹腔注射相同劑量的二甲基亞碸（DMSO）溶液；SB組於造模後第3、4、5天腹腔內注射SB431542（按4.2 mg/kg，0.5 mL/隻，溶於10%乙醇），其餘三組給予等體積10%乙醇。