氧化鈹致大鼠肺纖維化的實驗研究
發(fā)布時間：2018-11-27 15:11
【摘要】：目的本研究以BeO為染毒物質,復制大鼠肺纖維化動物模型,通過檢測實驗動物染毒Thy-1、TGFβ-smad2/3信號通路蛋白的表達,探討Thy-1、TGFβ1-smad2/3信號通路在鈹致肺纖維化中的意義,為后續(xù)研究提供依據(jù)。方法64只SPF級健康雄性SD大鼠,隨機分為陰性對照組(32只)、BeO染毒組(32只)。氧化鈹組氣管灌注BeO混懸液0.5ml,陰性對照組注入等量的無菌生理鹽水,處死時間為染毒后20、40、60和80 d四個時間點取材,分別進行以下實驗:1.取肺組織,制作石蠟切片并進行HE、Masson和免疫組化染色,同時制作電鏡切片。2.實時定量PCR(RT-PCR)法檢測COL-I,COL-III和α-SMA的基因表達水平。3.蛋白質印跡法(western-blot)檢測各組肺組織中COL-I,CLOL-III和α-SMA的蛋白水平。4.取肺組織制備肺組織勻漿,酶聯(lián)免疫法(ELISA)檢測Thy-1、TGFβ1、smad2、smad3、p-smad2、p-smad3的蛋白表達。結果1鼠肺組織解剖學形態(tài)和病理學的改變:大體觀察結果顯示:陰性對照組肺呈粉紅色,光滑質軟。BeO染毒后,隨染毒時間延長肺部損傷進行性加重,逐漸出現(xiàn)充血、體積增大、局部白色粟粒狀結節(jié)等,最終可見全肺結節(jié)及萎縮等。HE染色顯示:BeO染毒后20、40 d主要病理改變以炎性細胞浸潤為主,肺泡隔增厚;染毒后60、80 d肺泡腔縮小,部分肺泡結構消失和結構紊亂,局部可見纖維化結節(jié)且可見BeO粉塵顆粒,局部發(fā)生肺組織實變。Masson染色顯示:BeO染毒后支氣管及肺泡間隔可見藍色膠原沉積隨著時間的延長藍色膠原沉積明顯增加。電鏡結果顯示:BeO染毒20d和40d后呈現(xiàn)肺泡Ⅱ型上皮細胞胞漿嗜鋨小體排空;60d和80d后肺泡Ⅱ型上皮細胞胞漿嗜鋨小體嗜鋨小體致密,肺間質可見纖維束存在。2.BeO染毒組COL-I mRNA和蛋白表達升高(P0.05);BeO染毒組COL-I mRNA和蛋白隨著時間延長而增加(P0.05)。BeO染毒60、80 d COL-III mRNA和蛋白表達水平升高(P0.05);BeO染毒組COL-III mRNA和蛋白隨著時間延長而增加(P0.05)。BeO染毒后a-SMA mRNA表達升高(P0.05),BeO染毒組a-SMA蛋白表達高于同時間點的陰性對照組(P0.05);a-SMA mRNA隨著時間延長而增加(P0.05)。(3)BeO染毒TGFβ1表達升高(P0.05),TGFβ1隨著時間延長而增加(P0.05)。BeO染毒后smad2、smad3蛋白水平升高(P0.05),Smad3表達隨著時間延長而增加(P0.05);BeO染毒p-smad2、p-smad3表達升高(P0.05)并且隨著時間延長而增加。(4)BeO染毒后Thy-1表達降低,隨著時間延長而降低(P0.05)。結論(1)一次性非暴露式氣管內灌注質量濃度為10 g/L的BeO溶液0.5 mL可成功制備鈹化合物致SD大鼠肺纖維化模型。(2)BeO可引起肺組織中細胞外基質成分的沉積和成纖維細胞轉分化為肌成纖維細胞。(3)BeO誘導肺纖維化過程中TGFβ1-smad2/3信號通路被激活,TGFβ1-smad2/3信號通路參與肺纖維化的形成與發(fā)展。(4)BeO致肺纖維化過程中Thy-1的表達降低,導致天然的TGFβ1被激活。
[Abstract]:Objective to study the expression of Thy-1,TGF 尾-smad2/3 signaling pathway protein in rat pulmonary fibrosis model induced by BeO, and to explore the expression of Thy-1,. The significance of TGF 尾 1-smad2/3 signaling pathway in beryllium-induced pulmonary fibrosis provides a basis for further study. Methods 64 SPF grade healthy male SD rats were randomly divided into negative control group (32), BeO exposed rats). In beryllium oxide group, 0.5 ml of BeO suspension was infused into trachea, and the same amount of aseptic saline was injected into negative control group. The time of death was at 20 ~ 4060 and 80 days after exposure. The following experiments were carried out: 1. Lung tissue was taken, paraffin sections were made, HE,Masson and immunohistochemical staining were performed, and electron microscope sections were made. 2. The gene expression levels of COL-I,COL-III and 偽-SMA were detected by real-time quantitative PCR (RT-PCR). Protein levels of COL-I,CLOL-III and 偽-SMA in lung tissues were detected by Western blot (western-blot). 4. Lung homogenate was prepared from lung tissue and the protein expression of Thy-1,TGF 尾 1 smad2ssmad2 (p-smad2) p-smad3 was detected by enzyme-linked immunosorbent assay (ELISA). Results 1 the changes of lung anatomy and pathology: the results of gross observation showed that the lung of negative control group was pink, smooth and soft. After exposure to BeO, the lung injury was gradually aggravated with the prolongation of exposure time, and congestion gradually appeared. HE staining showed that the main pathological changes were inflammatory cell infiltration and alveolar septum thickening at 20 ~ 40 days after BeO exposure. At 6080 d after exposure, the alveolar cavity was reduced, part of the alveolar structure disappeared and the structure was disordered. Fibrosis nodules and BeO dust particles were observed in the area. Masson staining showed that blue collagen deposition increased significantly in bronchi and alveolar septum after BeO exposure with the prolongation of time. The results of electron microscope showed that the cytoplasmic osmiophilic bodies of alveolar type 鈪，
本文編號：2361240
[Abstract]:Objective to study the expression of Thy-1,TGF 尾-smad2/3 signaling pathway protein in rat pulmonary fibrosis model induced by BeO, and to explore the expression of Thy-1,. The significance of TGF 尾 1-smad2/3 signaling pathway in beryllium-induced pulmonary fibrosis provides a basis for further study. Methods 64 SPF grade healthy male SD rats were randomly divided into negative control group (32), BeO exposed rats). In beryllium oxide group, 0.5 ml of BeO suspension was infused into trachea, and the same amount of aseptic saline was injected into negative control group. The time of death was at 20 ~ 4060 and 80 days after exposure. The following experiments were carried out: 1. Lung tissue was taken, paraffin sections were made, HE,Masson and immunohistochemical staining were performed, and electron microscope sections were made. 2. The gene expression levels of COL-I,COL-III and 偽-SMA were detected by real-time quantitative PCR (RT-PCR). Protein levels of COL-I,CLOL-III and 偽-SMA in lung tissues were detected by Western blot (western-blot). 4. Lung homogenate was prepared from lung tissue and the protein expression of Thy-1,TGF 尾 1 smad2ssmad2 (p-smad2) p-smad3 was detected by enzyme-linked immunosorbent assay (ELISA). Results 1 the changes of lung anatomy and pathology: the results of gross observation showed that the lung of negative control group was pink, smooth and soft. After exposure to BeO, the lung injury was gradually aggravated with the prolongation of exposure time, and congestion gradually appeared. HE staining showed that the main pathological changes were inflammatory cell infiltration and alveolar septum thickening at 20 ~ 40 days after BeO exposure. At 6080 d after exposure, the alveolar cavity was reduced, part of the alveolar structure disappeared and the structure was disordered. Fibrosis nodules and BeO dust particles were observed in the area. Masson staining showed that blue collagen deposition increased significantly in bronchi and alveolar septum after BeO exposure with the prolongation of time. The results of electron microscope showed that the cytoplasmic osmiophilic bodies of alveolar type 鈪，
本文編號：2361240
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