内质网应激在视网膜缺血再灌注损伤中的作用

发布时间：2018-04-29 00:19

本文选题：视网膜 + 缺血再灌注损伤　；参考：《武汉大学》2012年博士论文

【摘要】：视网膜缺血再灌注(ischemia and reperfusion, I/R)损伤参与了包括急性闭角型青光眼和糖尿病视网膜病变在内的多种眼科疾病的发生和发展过程,而这些眼科疾病是导致人类视力损伤和失明的主要原因。视网膜I/R损伤能造成神经细胞和血管细胞的退化以及胶质细胞的损伤性激活,但其损伤机理还未完全研究清楚。本论文利用视网膜I/R损伤的小鼠模型,结合模拟缺血性损伤的细胞模型,通过RT-PCR、免疫共沉淀、蛋白质免疫印迹和免疫组织化学等多种手段,从分子水平、细胞水平和病理水平对视网膜I/R损伤的分子机理进行了研究。越来越多证据表明内质网应激(endoplasmic reticulum stress, ER stress)介导的细胞凋亡与I/R损伤有密切联系。我们首次发现,玻璃体腔注射ER stress诱导剂衣霉素能显著造成视网膜血管细胞退化。视网膜I/R损伤和衣霉素损伤这两种模型均造成了视网膜中ER stress标志性蛋白如CHOP的表达升高,特别是CHOP在血管细胞中的升高。白藜芦醇是一种具有抗炎症、抗氧化和抗肿瘤等多种活性的多酚类化合物。我们利用白藜芦醇来治疗经过视网膜I/R损伤和衣霉素损伤的小鼠,发现白藜芦醇(25mg/kg体重每天)能有效地降低两种损伤造成的ER stress升高,并且能抑制两种损伤造成的血管退化,但对I/R造成的神经性退化无影响。此部分结果表明ER stress参与了视网膜的血管退化过程。白藜芦醇的血管保护作用可能是由于它能抑制损伤造成的视网膜尤其是血管细胞中的ER stress。多聚ADP核糖聚合酶(poly(ADP-ribose) polymerase, PARP)已被证明在视网膜I/R损伤后过量激活。第一代PARP活性抑制剂能缓解视网膜I/R造成的神经损伤。在I/R损伤前或损伤后以灌胃方式给予第二代PARP活性抑制剂GPI15427(40mg/kg体重每天),能抑制损伤造成的神经退化,包括神经细胞缺失和细胞层厚度的降低。我们发现,GPI15427的神经保护作用可能是由于它能抑制I/R造成的视网膜尤其是神经细胞中ER stress相关蛋白CHOP和Bip的过量表达,但GPI15427对CHOP在血管细胞中的过表达和损伤造成的血管退化无影响。同时,我们还采用体外无糖无血清培养环境来模拟缺血性损伤。在该刺激下的视网膜米勒细胞系中,我们发现了多种ER stress信号通路相关蛋白的过量表达。此外,我们发现了mTOR信号通路在体内视网膜I/R损伤后激活,但抑制mTOR激活的药物雷帕霉素不能抑制损伤造成的神经和血管退化。4E-BP1作为mTOR下游磷酸化底物之一,其因水平、蛋白水平和蛋白磷酸化水平都在损伤后都显著上升,但4E-BP1在I/R损伤中的作用还不清楚。研究还发现视网膜I/R损伤造成了p53的过表达、乙酰化p53升高和去乙酰化酶SIRT1表达的下调等。本论文的研究结果表明,ER stress不仅参与了视网膜I/R损伤引起的神经退化,而且在损伤引起的血管退化中起着重要作用。白藜芦醇对血管保护作用和GPI15427对神经保护作用可能是通过分别抑制损伤引起的血管和神经细胞中的ER stress通路来实现的。此外,4E-BP1、p53和SIRT1的变化都可能参与由I/R引起的视网膜神经和血管退化,可以作为后续研究的内容。本项研究不仅为视网膜I/R损伤引起的神经和血管退化的病理发生提供了新的分子机理,而且为有此类病理损伤的疾病治疗提供了可能的药物作用靶点。
[Abstract]:Retinal ischemia-reperfusion (ischemia and reperfusion, I/R) injuries are involved in the occurrence and development of various ocular diseases, including acute angle closure glaucoma and diabetic retinopathy, which are the main causes of visual impairment and blindness in human beings. The retinal I/R damage can cause nerve cells and blood. The mechanism of the degeneration of the cells and the activation of the damage of the glial cells has not been fully studied. In this paper, the mouse model of retinal I/R damage, combined with the cell model of the simulated ischemic damage, through RT-PCR, immunoprecipitation, protein immunoblotting and immunohistochemistry, from the molecular level, cell The molecular mechanism of retinal I/R damage was studied at the level and pathological level.
There is increasing evidence that endoplasmic reticulum stress (ER stress) mediated apoptosis is closely related to I/R damage. For the first time, we found that intravitreal injection of ER stress inducer, ycomycin, can significantly cause retinal vascular cell degeneration. The two models of retinal I/ R damage and mycophenin damage are all caused by these models. The expression of ER stress marker protein, such as CHOP, is elevated in the retina, especially the increase of CHOP in vascular cells. Resveratrol is a polyphenolic compound with anti-inflammatory, antioxidation and antitumor activity. We use resveratrol to treat mice that have been injured by I/R and mycophenin, and find resveratrol ( 25mg/kg weight per day) effectively reduces the increase of ER stress caused by two injuries and inhibits the vascular degeneration caused by two injuries, but has no effect on the neurodegeneration caused by I/R. This part suggests that ER stress is involved in the vascular degeneration of the retina. The protective effect of resveratrol may be due to its inhibition ER stress. in damaged retina, especially in vascular cells.
ADP ribose polymerase (poly (ADP-ribose) polymerase, PARP) has been proved to be excessively activated after retinal I/R damage. The first generation PARP active inhibitor can relieve the nerve damage caused by retinal I/R. The second generation PARP active inhibitor, GPI15427 (40mg/kg weight every day), can be inhibited before or after the damage of I/R injury. Neurodegeneration, including the loss of nerve cells and the decrease of cell layer thickness, we have found that the neuroprotective effect of GPI15427 may be due to the inhibition of the overexpression of the ER stress related protein CHOP and Bip in the retina, especially in the neurons, caused by I/R, but the over expression and damage of CHOP in vascular cells There is no effect on vascular degeneration.
At the same time, we also used in vitro sugar free and serum-free culture environment to simulate ischemic injury. In the retina Miller cell line, we found a variety of ER stress signaling related protein overexpression. In addition, we found that the mTOR signaling pathway activates after the I/R damage of the retina membrane in the body, but inhibits the mTOR activated drug. Rapamycin can not inhibit the neurovascular and vascular degradation caused by.4E-BP1 as one of the phosphorylation substrates in the downstream mTOR. Its level, protein level and protein phosphorylation level are all significantly increased after injury, but the role of 4E-BP1 in I/R damage is not clear. The study also found that the I/R damage of the retina caused the overexpression of p53 and the acetylation of P. 53 increase and down regulation of deacetylase SIRT1 expression.
The results of this study show that ER stress not only participates in the neurodegeneration caused by I/R damage in the retina, but also plays an important role in the vascular degeneration caused by injury. The protective effect of resveratrol on blood vessels and the protective effect of GPI15427 on the nerve may be through the ER stress in the vascular and nerve cells caused by the inhibition of damage. In addition, changes in 4E-BP1, p53 and SIRT1 may be involved in the degeneration of retinal nerve and blood vessels caused by I/R, which can be used as a follow-up study. This study not only provides a new subdivision mechanism for the pathological changes of nerve and vascular degeneration caused by I/R damage in the retina, but also for the treatment of diseases with such pathological damage. Therapy provides a possible target for drug action.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1

【参考文献】