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论文题名(中文):

 MIF、p-ERK、cyclinD1在肺动脉高压肉鸡肺脏中的分布与表达    

作者:

 李浩运    

学号:

 20132316    

保密级别:

 公开    

论文语种:

 chi    

学科代码:

 S852.33    

学生类型:

 硕士    

学位:

 农学硕士    

学校:

 山西农业大学    

院系:

 动物科技学院    

专业:

 基础兽医学    

研究方向:

 炎症分子的作用机制    

第一导师姓名:

 王文魁    

第一导师学校:

 山西农业大学    

论文完成日期:

 2016-04-15    

论文答辩日期:

 2016-6-12    

论文题名(外文):

 The expression and distribution of MIF,p-ERK and cyclinD1 in the lung of broilers with pulmonary hypertension    

关键词(中文):

 肉鸡 肺动脉高压 MIF p-ERK/ERK cyclinD1    

关键词(外文):

 broiler pulmonary hypertension MIF ERK/p-ERK cyclinD1    

论文文摘(中文):

【目的】肉鸡肺动脉高压(PH)严重影响了我国肉鸡产业的发展。该病发生发展过程的重要环节是以肺动脉平滑肌细胞增殖为主的肺动脉重构。细胞周期蛋白D1是调控细胞增殖的关键因子(之一),该因子是否参与患病肉鸡肺动脉平滑肌增殖尚未见报道。据报道,激活cyclinD1的信号通路主要有细胞外信号调节激酶(ERK);结合我们前期工作,发现肺动脉高压肉鸡肺脏中巨噬细胞移动抑制因子(MIF)表达量显著高于健康肉鸡,说明MIF参与了肉鸡PH的发病过程。本试验目的就是要理清三者与肉鸡PH发病的关系。

【方法】PH组为3-4周龄左右临床型肺动脉高压肉鸡,正常组为同群同日龄健康肉鸡,均由山西省大象集团AA肉鸡养殖场提供。通过HE染色观察PH组与正常组肺组织血管形态的区别,利用免疫组织化学及免疫印迹技术,对肉鸡肺脏组织MIFp-ERKcyclinD1三种蛋白进行定位及半定量分析,并对比PH组与正常组的表达差异。

【结果】HE染色结果显示,正常组肉鸡肺脏组织结构清晰、完整,肺血管内膜、中膜形态正常,无明显增厚现象。与正常组相比,PH组出现动脉内膜及中膜增厚的现象,其管腔变窄,管壁较正常组显著增厚。

免疫组化结果显示,在肉鸡肺动脉壁上,MIFp-ERKcyclinD1三个蛋白均呈阳性表达。其中,MIF表达于血管内皮细胞和中膜平滑肌细胞胞质中;p-ERKcyclinD1定位于血管中膜平滑肌细胞胞质中。

Western-blot结果显示,与正常组相比,肺动脉高压组肉鸡肺脏MIFCyclin D1表达量显著升高(P0.01);肺动脉高压组磷酸化ERK(p-ERK)较正常组显著增多(P0.01),表明ERK通路处于高度激活状态。

【结论】肉鸡PH发病过程中,中小型肺动脉中膜增厚,其原因可能与肺脏MIF表达量升高,ERK信号通路高度活化,cyclinD1表达量升高等变化有关。
文摘(外文):

[Purpose] Pulmonary hypertension (PH) is one of major diseases in the broiler breeding industry. A significant tache in the development of PH is the remodeling of pulmonary artery, which gives priority to the proliferation of pulmonary vascular smooth muscle cells(PVSMCs). CyclinD1 is a key factor in the regulation of cell proliferation, and if this factor is involved in the PVSMCs proliferation of broilers with PH has not been reported. Therefore, we conducted this study. According to the reports, extracellular signal-regulated kinase (ERK) is the major signal pathway to regulate the expression of cyclinD1. In addition, our previous work has shown that the expression of macrophage migration inhibition factor (MIF) in broiler lungs with pulmonary hypertension is significantly higher than those in healthy broilers, which means that MIF has involved in the development of PH in broilers. This experiment is aiming to clarify the relationship among these three proteins on the pathogenesis of PH in broiler, which is to say whether MIF could induce pulmonary artery wall thickening by activating ERK1/2 pathway to upregulate the expression of cyclinD1 and thereby promoting the PVSMCs proliferation.

[Methods] The PH group was about 30 day-old broilers with clinical pulmonary hypertension, while the normal was healthy broilers at the same age. The difference of lung vascular morphology between PH group and healthy group was observed by HE staining. The location and expression of MIF, p-ERK and cyclinD1 proteins were detected by Immunohisto-chemistry and western-blotting, and the results between two groups was compared.

[Results] The results of HE staining showed that the structure of normal lung could be observed clearly and completely, without leakage and inflammatory cell infiltration. Compared with normal group, the PH group showed arterial intima and media thickening, luminal narrowing and more pulmonary inflammatory cell infiltration around pulmonary vascular observed.

The results of Immunohistochemistry showed that positive reaction of MIF, p-ERK and cyclinD1 was observed on the pulmonary vascular walls, in which the expression of MIF located in the cytoplasm of vascular endothelial cells and smooth muscle cells; p-ERK and cyclinD1 located in the cytoplasm of vascular smooth muscle cells.

The results of Western-blotting showed that compared with the normal group, the expression of MIF and cyclinD1 in PH group increased significantly (P<0.01), and the phosphorylation of ERK (p-ERK) was significantly upregulated (P<0.01), which indicated ERK pathway was highly activated.

[Conclusion] The pulmonary artery media membrane thickening appeared during the development of PH in broilers, which might be related to the increased expression of MIF in lungs, ERK signaling pathway highly activated, and upregulation of cyclinD1.

论文目录:

目录


摘要.................................................................................................. - 1 -


前言................................................................................................... - 2 -


文献综述............................................................................................ - 3 -


1 肉鸡养殖业现状.......................................................................... - 3 -


2 肉鸡肺动脉高压.......................................................................... - 3 -


2.1 流行病学.......................................................................... - 4 -


2.2 发病特点.......................................................................... - 4 -


2.3 诊断标准.......................................................................... - 5 -


2.4 组织学变化....................................................................... - 5 -


2.5 发病机制.......................................................................... - 5 -


2.6 PH的防治......................................................................... - 8 -


3 CyclinD1与肺动脉高压................................................................ - 9 -


3.1 CyclinD1的基本结构......................................................... - 9 -


3.2 CyclinD1与细胞周期......................................................... - 9 -


3.3 CyclinD1与癌症.............................................................. - 11 -


3.4 CyclinD1与血管疾病........................................................ - 11 -


3.5 CyclinD1与ERK、MIF....................................................... - 11 -


试验一 肉鸡肺脏的HE染色与MIF、p-ERK、cyclinD1的免疫组织化学检测.... - 13 -


1动物........................................................................................ - 13 -


2 试验仪器.................................................................................. - 13 -


3 试验试剂及配方......................................................................... - 13 -


3.1 主要试验试剂.................................................................. - 13 -


3.2 抗体.............................................................................. - 13 -


3.3 主要试剂配置方法............................................................ - 13 -


4 试验步骤.................................................................................. - 14 -


4.1 石蜡切片的制作............................................................... - 14 -


4.2 HE染色.......................................................................... - 14 -


4.3 免疫组织化学试验............................................................ - 15 -


4.4 图片处理及数据分析软件与方法.......................................... - 15 -


5 结果与分析............................................................................... - 16 -


5.1 肺脏组织学变化............................................................... - 16 -


5.2 MIF、p-ERK、Cyclin D1在肉鸡肺动脉中的定位.................... - 17 -


6 小结........................................................................................ - 18 -


试验二 肉鸡肺脏中MIFp-ERKcyclinD1的免疫印迹检测........................ - 19 -


1 动物........................................................................................ - 19 -


2 试验仪器.................................................................................. - 19 -


3 试验试剂及配方......................................................................... - 19 -


3.1主要试验试剂................................................................... - 19 -


3.3主要试剂配制方法............................................................. - 20 -


4 试验步骤.................................................................................. - 20 -


4.1 肉鸡组织抗体可用性检测................................................... - 20 -


4.2 组织蛋白的提取............................................................... - 21 -


4.3 BCA法测定蛋白浓度.......................................................... - 21 -


4.4 SDS-聚丙烯酰胺凝胶电泳................................................... - 21 -


4.5 转膜.............................................................................. - 22 -


4.6 封闭与杂交..................................................................... - 22 -


4.7 图像处理与数据分析......................................................... - 23 -


5 试验结果.................................................................................. - 23 -


5.1 抗体对于肉鸡组织可用性检测............................................. - 23 -


5.2 MIF、p-ERK/ERK、cyclinD1在肉鸡肺脏表达定量................... - 24 -


6 小结........................................................................................ - 25 -


讨论................................................................................................ - 26 -


结论................................................................................................ - 29 -


参考文献.......................................................................................... - 30 -


............................................................................................... - 35 -

中图分类号:

 S852.33    

馆藏号:

 Y7943    

开放日期:

 2016-04-15    

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