氧化铍致大鼠肺纤维化的实验研究
发布时间：2018-11-27 15:11
【摘要】：目的本研究以BeO为染毒物质,复制大鼠肺纤维化动物模型,通过检测实验动物染毒Thy-1、TGFβ-smad2/3信号通路蛋白的表达,探讨Thy-1、TGFβ1-smad2/3信号通路在铍致肺纤维化中的意义,为后续研究提供依据。方法64只SPF级健康雄性SD大鼠,随机分为阴性对照组(32只)、BeO染毒组(32只)。氧化铍组气管灌注BeO混悬液0.5ml,阴性对照组注入等量的无菌生理盐水,处死时间为染毒后20、40、60和80 d四个时间点取材,分别进行以下实验:1.取肺组织,制作石蜡切片并进行HE、Masson和免疫组化染色,同时制作电镜切片。2.实时定量PCR(RT-PCR)法检测COL-I,COL-III和α-SMA的基因表达水平。3.蛋白质印迹法(western-blot)检测各组肺组织中COL-I,CLOL-III和α-SMA的蛋白水平。4.取肺组织制备肺组织匀浆,酶联免疫法(ELISA)检测Thy-1、TGFβ1、smad2、smad3、p-smad2、p-smad3的蛋白表达。结果1鼠肺组织解剖学形态和病理学的改变:大体观察结果显示:阴性对照组肺呈粉红色,光滑质软。BeO染毒后,随染毒时间延长肺部损伤进行性加重,逐渐出现充血、体积增大、局部白色粟粒状结节等,最终可见全肺结节及萎缩等。HE染色显示:BeO染毒后20、40 d主要病理改变以炎性细胞浸润为主,肺泡隔增厚;染毒后60、80 d肺泡腔缩小,部分肺泡结构消失和结构紊乱,局部可见纤维化结节且可见BeO粉尘颗粒,局部发生肺组织实变。Masson染色显示:BeO染毒后支气管及肺泡间隔可见蓝色胶原沉积随着时间的延长蓝色胶原沉积明显增加。电镜结果显示:BeO染毒20d和40d后呈现肺泡Ⅱ型上皮细胞胞浆嗜锇小体排空;60d和80d后肺泡Ⅱ型上皮细胞胞浆嗜锇小体嗜锇小体致密,肺间质可见纤维束存在。2.BeO染毒组COL-I mRNA和蛋白表达升高(P0.05);BeO染毒组COL-I mRNA和蛋白随着时间延长而增加(P0.05)。BeO染毒60、80 d COL-III mRNA和蛋白表达水平升高(P0.05);BeO染毒组COL-III mRNA和蛋白随着时间延长而增加(P0.05)。BeO染毒后a-SMA mRNA表达升高(P0.05),BeO染毒组a-SMA蛋白表达高于同时间点的阴性对照组(P0.05);a-SMA mRNA随着时间延长而增加(P0.05)。(3)BeO染毒TGFβ1表达升高(P0.05),TGFβ1随着时间延长而增加(P0.05)。BeO染毒后smad2、smad3蛋白水平升高(P0.05),Smad3表达随着时间延长而增加(P0.05);BeO染毒p-smad2、p-smad3表达升高(P0.05)并且随着时间延长而增加。(4)BeO染毒后Thy-1表达降低,随着时间延长而降低(P0.05)。结论(1)一次性非暴露式气管内灌注质量浓度为10 g/L的BeO溶液0.5 mL可成功制备铍化合物致SD大鼠肺纤维化模型。(2)BeO可引起肺组织中细胞外基质成分的沉积和成纤维细胞转分化为肌成纤维细胞。(3)BeO诱导肺纤维化过程中TGFβ1-smad2/3信号通路被激活,TGFβ1-smad2/3信号通路参与肺纤维化的形成与发展。(4)BeO致肺纤维化过程中Thy-1的表达降低,导致天然的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 伪-SMA were detected by real-time quantitative PCR (RT-PCR). Protein levels of COL-I,CLOL-III and 伪-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，
本文编号：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 伪-SMA were detected by real-time quantitative PCR (RT-PCR). Protein levels of COL-I,CLOL-III and 伪-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，
本文编号：2361240
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