内质网应激在特发性肺纤维化发病机制中的作用

发布时间：2018-03-15 23:33

本文选题：内质网应激　切入点：Grp78　出处：《北京协和医学院》2013年博士论文　论文类型：学位论文

【摘要】：研究背景特发性肺纤维化(idiopathic pulmonary fibrosis, IPF)是一种慢性进展性破坏性的肺实质疾病,以肺泡结构逐渐破坏被纤维组织取代为特点,临床表现为进展性呼吸困难及肺功能下降。IPF约占所有间质性肺病的20-30%,目前美国及欧盟患者总共约500,000人,IPF预后较差,中位生存期约2.8-4.2年,无有效的治疗方法,对于病情晚期的患者肺移植为治疗的唯一选择。IPF发生发展的病因目前仍未可知,长久以来认为慢性炎症为此病发生发展的基础,但类固醇激素及免疫抑制剂治疗作用有限甚至无效。因此,慢性肺泡上皮细胞损伤继而引发肺泡异常修复这一假说逐渐成为研究的热点,肺泡上皮细胞超微结构研究发现疾病早期即有肺泡上皮细胞死亡,支持慢性肺泡上皮细胞损伤继而引发肺泡异常修复假说。在病变肺组织中不仅成纤维细胞灶表面覆盖的增生的Ⅱ型肺泡上皮细胞凋亡增加,外观几乎正常的肺组织Ⅱ型肺泡上皮细胞凋亡也增加,与此相一致的是,阻断凋亡通路的传导可减轻博来霉素诱导肺纤维化模型的肺纤维化程度。干扰内质网的蛋白质加工折叠功能可引起错误折叠蛋白在内质网聚积,从而引起内质网应激(endoplasmic reticulum stress, ERS)和未折叠蛋白反应(unfolded protein response, UPR)激活。有些疾病与蛋白错误折叠相关,近几年研究发现IPF患者肺泡上皮细胞内质网应激和未折叠蛋白反应的活化明显增强,表明内质网应激在IPF的发生发展中可能发挥作用。内质网应激通过内质网膜上3个跨膜蛋白PERK、ATF6和IRE1诱导3条独立的ERS信号通路的转导。本研究通过观察内质网应激通路激活情况,探讨内质网应激在IPF发生发展中的作用。研究目的观察内质网应激通路激活情况,明确内质网应激在IPF发生发展中的作用。实验方法 1.采用免疫组织化学的方法观察IPF患者及博来霉素诱导小鼠肺纤维化肺组织Grp78、CHOP蛋白的表达,观察内质网应激通路激活情况； 2.衣霉素(tunicamycin, TM)是一种蛋白糖基化抑制剂,可诱导细胞发生内质网应激。本试验通过衣霉素诱导A549细胞应激,用Western blot法检测此过程中Grp78、CHOP蛋白表达的变化,通过体外细胞培养研究观察内质网应激通路激活情况,初步探讨细胞凋亡情况。实验结果 1.10例IPF患者肺组织Grp78、CHOP蛋白均表达阳性；正常对照组中16例正常肺组织中12.5%(2例)Grp78蛋白表达阳性,18.8%(3例)CHOP蛋白表达阳性。 2.4例博来霉素诱导小鼠肺纤维化肺组织Grp78、CHOP蛋白均表达阳性；4例正常对照组肺组织Grp78、CHOP蛋白均未见表达。 3.衣霉素引起内质网应激未折叠蛋白反应分子伴侣Grp78及凋亡通路蛋白CHOP上调,提示衣霉素可诱导A549细胞内质网应激激活,从未折叠蛋白反应转向前凋亡反应；根据CHOP表达随时间及浓度逐渐增加,推断衣霉素可诱导细胞凋亡,且呈依赖性及浓度依赖性。实验结论内质网应激通过诱导肺泡上皮细胞凋亡在IPF的发生发展中发挥作用。
[Abstract]:Research background
Idiopathic pulmonary fibrosis (idiopathic pulmonary, fibrosis, IPF) is a chronic progressive destructive lung disease, with the gradual destruction of the alveolar structure was replaced by fibrous tissue characteristics, clinical manifestations of progressive dyspnea and lung function decline.IPF about all interstitial lung disease in 20-30% patients, the United States and the European Union a total of about 500000 people, IPF and poor prognosis, the median survival period of about 2.8-4.2 years, no effective treatment, patients with lung transplantation for patients with advanced diseases only choice for.IPF development is still unknown, have long thought that the basic for the occurrence and development of chronic inflammatory disease, but the treatment effects of steroids and immune Limited or ineffective inhibitor. Therefore, chronic damage of alveolar epithelial cells leading to abnormal alveolar repair this hypothesis has gradually become the research hotspot, alveolar epithelial cell ultrastructure The study found that the disease in early stage alveolar epithelial cell death, chronic damage of alveolar epithelial cells leading to abnormal alveolar repair hypothesis. Not only fibroblasts proliferate range covering the surface of the apoptosis of type II alveolar epithelial cells increased in lung tissue lesions, the appearance of almost normal lung tissue of type II alveolar epithelial cell apoptosis also increased, phase consistent with this, blocking apoptosis pathways can alleviate bleomycin induced pulmonary fibrosis model of pulmonary fibrosis. Processing of protein folding function can cause disturbance of endoplasmic reticulum, endoplasmic reticulum accumulation of misfolded proteins, and cause endoplasmic reticulum stress (endoplasmic reticulum, stress, ERS) and the unfolded protein response (unfolded protein response, UPR) activation and protein misfolding diseases. Some related research in recent years found that IPF patients with alveolar epithelial cells of endoplasmic reticulum stress and unfolded The activation stack protein response was significantly enhanced, showed that the endoplasmic reticulum stress in the occurrence and development of IPF may play a role of endoplasmic reticulum stress through endoplasmic reticulum 3 transmembrane protein PERK, 3 independent ERS signal transduction pathway induced by ATF6 and IRE1. This study observed the endoplasmic reticulum stress pathway of endoplasmic reticulum, stress in the development of IPF.
research objective
To observe the activation of endoplasmic reticulum stress pathway and to clarify the role of endoplasmic reticulum stress in the development of IPF.
Experimental method
1. immunohistochemical method was used to observe the expression of Grp78 and CHOP protein in lung tissues of patients with IPF and bleomycin induced pulmonary fibrosis, and to observe the activation of endoplasmic reticulum stress pathway.
2. tunicamycin (tunicamycin, TM) is a kind of protein glycosylation inhibitor, can induce endoplasmic reticulum stress. The tunicamycin induced stress in A549 cells, detection of Grp78 in this process by using Western blot method, the changes of the expression of CHOP protein, in vitro cell culture research on endoplasmic reticulum stress pathway the activation, apoptosis.
experimental result
The expression of Grp78 and CHOP protein was positive in 1.10 cases of IPF. In the normal control group, 12.5% (2 cases) had positive Grp78 protein expression in 16 cases of normal lung tissues, and 18.8% (3 cases) had positive expression of CHOP protein.
In 2.4 cases of bleomycin induced lung fibrosis in mice, Grp78, CHOP protein expression was positive, and 4 cases of normal control group Grp78, no expression of CHOP protein.
3. tunicamycin induced ER stress unfolded protein response molecular chaperone Grp78 and apoptosis protein CHOP is upregulated, suggesting that tunicamycin induced endoplasmic reticulum stress activated A549 cells, has unfolded protein response before turning apoptosis reaction; according to the expression of CHOP with time and concentration gradually increased, that tunicamycin induces apoptosis. And a dependent and concentration dependent.
empirical conclusion
Endoplasmic reticulum stress plays a role in the development of IPF by inducing apoptosis of alveolar epithelial cells.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563.9

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