本文選題：視網(wǎng)膜 + 缺血再灌注損傷　； 參考：《武漢大學(xué)》2012年博士論文

【摘要】：視網(wǎng)膜缺血再灌注(ischemia and reperfusion, I/R)損傷參與了包括急性閉角型青光眼和糖尿病視網(wǎng)膜病變?cè)趦?nèi)的多種眼科疾病的發(fā)生和發(fā)展過(guò)程,而這些眼科疾病是導(dǎo)致人類視力損傷和失明的主要原因。視網(wǎng)膜I/R損傷能造成神經(jīng)細(xì)胞和血管細(xì)胞的退化以及膠質(zhì)細(xì)胞的損傷性激活,但其損傷機(jī)理還未完全研究清楚。本論文利用視網(wǎng)膜I/R損傷的小鼠模型,結(jié)合模擬缺血性損傷的細(xì)胞模型,通過(guò)RT-PCR、免疫共沉淀、蛋白質(zhì)免疫印跡和免疫組織化學(xué)等多種手段,從分子水平、細(xì)胞水平和病理水平對(duì)視網(wǎng)膜I/R損傷的分子機(jī)理進(jìn)行了研究。 越來(lái)越多證據(jù)表明內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress, ER stress)介導(dǎo)的細(xì)胞凋亡與I/R損傷有密切聯(lián)系。我們首次發(fā)現(xiàn),玻璃體腔注射ER stress誘導(dǎo)劑衣霉素能顯著造成視網(wǎng)膜血管細(xì)胞退化。視網(wǎng)膜I/R損傷和衣霉素?fù)p傷這兩種模型均造成了視網(wǎng)膜中ER stress標(biāo)志性蛋白如CHOP的表達(dá)升高,特別是CHOP在血管細(xì)胞中的升高。白藜蘆醇是一種具有抗炎癥、抗氧化和抗腫瘤等多種活性的多酚類化合物。我們利用白藜蘆醇來(lái)治療經(jīng)過(guò)視網(wǎng)膜I/R損傷和衣霉素?fù)p傷的小鼠,發(fā)現(xiàn)白藜蘆醇(25mg/kg體重每天)能有效地降低兩種損傷造成的ER stress升高,并且能抑制兩種損傷造成的血管退化,但對(duì)I/R造成的神經(jīng)性退化無(wú)影響。此部分結(jié)果表明ER stress參與了視網(wǎng)膜的血管退化過(guò)程。白藜蘆醇的血管保護(hù)作用可能是由于它能抑制損傷造成的視網(wǎng)膜尤其是血管細(xì)胞中的ER stress。 多聚ADP核糖聚合酶(poly(ADP-ribose) polymerase, PARP)已被證明在視網(wǎng)膜I/R損傷后過(guò)量激活。第一代PARP活性抑制劑能緩解視網(wǎng)膜I/R造成的神經(jīng)損傷。在I/R損傷前或損傷后以灌胃方式給予第二代PARP活性抑制劑GPI15427(40mg/kg體重每天),能抑制損傷造成的神經(jīng)退化,包括神經(jīng)細(xì)胞缺失和細(xì)胞層厚度的降低。我們發(fā)現(xiàn),GPI15427的神經(jīng)保護(hù)作用可能是由于它能抑制I/R造成的視網(wǎng)膜尤其是神經(jīng)細(xì)胞中ER stress相關(guān)蛋白CHOP和Bip的過(guò)量表達(dá),但GPI15427對(duì)CHOP在血管細(xì)胞中的過(guò)表達(dá)和損傷造成的血管退化無(wú)影響。 同時(shí),我們還采用體外無(wú)糖無(wú)血清培養(yǎng)環(huán)境來(lái)模擬缺血性損傷。在該刺激下的視網(wǎng)膜米勒細(xì)胞系中,我們發(fā)現(xiàn)了多種ER stress信號(hào)通路相關(guān)蛋白的過(guò)量表達(dá)。此外,我們發(fā)現(xiàn)了mTOR信號(hào)通路在體內(nèi)視網(wǎng)膜I/R損傷后激活,但抑制mTOR激活的藥物雷帕霉素不能抑制損傷造成的神經(jīng)和血管退化。4E-BP1作為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損傷后都顯著上升,但4E-BP1在I/R損傷中的作用還不清楚。研究還發(fā)現(xiàn)視網(wǎng)膜I/R損傷造成了p53的過(guò)表達(dá)、乙�；痯53升高和去乙�；窼IRT1表達(dá)的下調(diào)等。 本論文的研究結(jié)果表明,ER stress不僅參與了視網(wǎng)膜I/R損傷引起的神經(jīng)退化,而且在損傷引起的血管退化中起著重要作用。白藜蘆醇對(duì)血管保護(hù)作用和GPI15427對(duì)神經(jīng)保護(hù)作用可能是通過(guò)分別抑制損傷引起的血管和神經(jīng)細(xì)胞中的ER stress通路來(lái)實(shí)現(xiàn)的。此外,4E-BP1、p53和SIRT1的變化都可能參與由I/R引起的視網(wǎng)膜神經(jīng)和血管退化,可以作為后續(xù)研究的內(nèi)容。本項(xiàng)研究不僅為視網(wǎng)膜I/R損傷引起的神經(jīng)和血管退化的病理發(fā)生提供了新的分子機(jī)理,而且為有此類病理?yè)p傷的疾病治療提供了可能的藥物作用靶點(diǎn)。
[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.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R774.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 鄭廣瑛,張成,李志剛;Early activation of caspase-1 after retinal ischemia and reperfusion injury in mice[J];Chinese Medical Journal;2004年05期

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本文編號(hào)：1817532