本文選題：年齡相關(guān)性黃斑變性 + 全反式視黃醛　； 參考：《浙江大學(xué)》2015年博士論文

【摘要】：年齡相關(guān)性視網(wǎng)膜黃斑變性(age-related macular degeneration, AMD)是一種嚴(yán)重影響老年人生活質(zhì)量的致盲性眼病。大量的研究證明：由于視網(wǎng)膜中全反式視黃醛(all-trans-retinal, atRAL)清除代謝障礙或急性過(guò)量光暴露而導(dǎo)致atRAL在視網(wǎng)膜中的過(guò)量累積可能是視網(wǎng)膜黃斑變性的關(guān)鍵原因之一。atRAL對(duì)視網(wǎng)膜細(xì)胞損傷的分子機(jī)制有待進(jìn)一步闡明,因而本課題選擇對(duì)維持視網(wǎng)膜正常生理功能非常重要的視網(wǎng)膜色素上皮(retinal pigment epithelium, RPE)細(xì)胞,在體外水平上研究了atRAL對(duì)該細(xì)胞的毒性損傷的機(jī)制,并初步探索了RPE細(xì)胞對(duì)atRAL的代謝消除方式。AMD病人眼底累積有大量的脂褐質(zhì)色素成分——類似色素二聚體(bisretinoids),而人們對(duì)這些色素成分在AMD發(fā)病中所起到的作用目前尚無(wú)統(tǒng)一認(rèn)識(shí)。大量的研究證實(shí)類視色素二聚體產(chǎn)物具有一定的生物活性,因而其在視網(wǎng)膜中的過(guò)量積聚可能會(huì)一定程度促進(jìn)視網(wǎng)膜變性的進(jìn)展。視網(wǎng)膜中脂褐質(zhì)色素的組成極其復(fù)雜,對(duì)這些成分的結(jié)構(gòu)解析、活性研究與其生物合成通路的確定有助于我們?cè)黾訉?duì)其存在意義及atRAL在視網(wǎng)膜中代謝的認(rèn)識(shí)。因而本論文最后一部分對(duì)視網(wǎng)膜中一個(gè)新穎的atRAL代謝成分進(jìn)行了結(jié)構(gòu)解析和活性研究。1.內(nèi)質(zhì)網(wǎng)應(yīng)激促進(jìn)了全反式視黃醛誘導(dǎo)的視網(wǎng)膜色素上皮細(xì)胞凋亡過(guò)度累積的內(nèi)源性atRAL會(huì)導(dǎo)致RPE細(xì)胞和感光細(xì)胞的大量凋亡,這被認(rèn)為在AMD和青少年型視網(wǎng)膜黃斑變性疾病Stargardt病的發(fā)病中起到一定的作用。在本章中,我們發(fā)現(xiàn)人RPE細(xì)胞系A(chǔ)RPE-19能夠耐受低濃度的atRAL,但是較高濃度的atRAL對(duì)RPE細(xì)胞表現(xiàn)出損傷作用,引起細(xì)胞大量凋亡。進(jìn)一步的研究發(fā)現(xiàn),atRAL的處理使RPE細(xì)胞中活性氧(ROS)顯著升高,并上調(diào)Nrf2, HO-1和y-GCSh的表達(dá),引起細(xì)胞氧化應(yīng)激反應(yīng)。亞細(xì)胞定位發(fā)現(xiàn)細(xì)胞中的ROS大量定位于線粒體和內(nèi)質(zhì)網(wǎng)上。atRAL上調(diào)了BiP的蛋白表達(dá)水平,并能濃度依賴性上調(diào)ATF4.CHOP和GADD34的表達(dá)水平。以上結(jié)果表明,atRAL引起了RPE細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激,激活了PERK-eIF2α-ATF4通路。進(jìn)一步研究發(fā)現(xiàn),內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑salubrinal能夠一定程度緩解atRAL所致RPE細(xì)胞死亡,且抗氧化劑N-乙酰半胱氨酸(NAC)能有效抑制RPE細(xì)胞死亡及內(nèi)質(zhì)網(wǎng)應(yīng)激的激活。最后,我們發(fā)現(xiàn)經(jīng)atRAL處理后,RPE細(xì)胞的線粒體膜電位明顯降低,激活了caspase 3和PARP。綜上,我們發(fā)現(xiàn)過(guò)度累積的atRAL可誘導(dǎo)RPE細(xì)胞內(nèi)產(chǎn)生過(guò)量的ROS并激活細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激,內(nèi)質(zhì)網(wǎng)應(yīng)激加重了RPE細(xì)胞的線粒體功能障礙,最終促進(jìn)了細(xì)胞的凋亡。2.視網(wǎng)膜色素上皮細(xì)胞對(duì)全反式視黃醛的代謝及全反式視黃醛二聚體形成意義的探索在神經(jīng)視網(wǎng)膜層過(guò)度積聚的atRAL最終會(huì)進(jìn)入RPE層,從而能夠?qū)PE細(xì)胞造成顯著影響。在本章中,我們發(fā)現(xiàn)避光條件下RPE細(xì)胞和atRAL孵育后,atRAL可以被代謝為全反式視黃醇(all-trans-retinol,atROL)和全反式視黃醛二聚體(all-trans-retinal dimer,atRAL-dimer)、A2E及其異構(gòu)體等產(chǎn)物,其中atRAL-dimer的量顯著高于A2E的量。同野生型小鼠相比,A2E及其異構(gòu)體、atRAL-dimer的累積量在RDH8-/-ABCA4-/-小鼠眼球中顯著升高,但atRAL-dimer的累積量及升高幅度均顯著高于A2E及其異構(gòu)體。我們發(fā)現(xiàn)同等條件下atRAL-dimer對(duì)RPE細(xì)胞活力、細(xì)胞線粒體的膜電位的抑制作用顯著低于atRAL,其誘導(dǎo)細(xì)胞活性氧產(chǎn)生及氧化應(yīng)激的相關(guān)基因Nrf2,HO-1和γGCSh表達(dá)的能力顯著弱于atRAL.綜上,我們發(fā)現(xiàn)在RPE細(xì)胞中的代謝atRAL途徑包括還原為atROL通路和以atRAL-dimer為代表的類視色素二聚體生成通路；體內(nèi)外的結(jié)果均表明,atRAL過(guò)量時(shí)易于在細(xì)胞內(nèi)累積atRAL-dimer;atRAL轉(zhuǎn)變?yōu)閍tRAL-dimer后,毒性顯著降低,因此atRAL-dimer的生成參與緩解atRAL對(duì)RPE細(xì)胞的毒性損傷。3.新穎視網(wǎng)膜脂褐質(zhì)色素成分iisoA2E的發(fā)現(xiàn)及其對(duì)視網(wǎng)膜色素上皮細(xì)胞的影響大量的研究表明,在RPE細(xì)胞中過(guò)度累積的脂褐質(zhì)成分與AMD及Stargardt病的發(fā)生可能存在一定的關(guān)聯(lián)。RPE脂褐質(zhì)中存在大量的具有自發(fā)熒光特性的類視色素二聚體成分,該類成分在視網(wǎng)膜變性中的作用尚需進(jìn)一步闡明。在本章,我們從視網(wǎng)膜中分離鑒定出一個(gè)新穎的脂褐質(zhì)色素成分iisoA2E。我們檢測(cè)到iisoA2E廣泛存在于小鼠、牛、豬及人的眼球或RPE/脈絡(luò)膜組織有機(jī)溶劑提取物中。在體外利用atRAL和乙醇胺反應(yīng)能夠制得iisoA2E,使得我們能夠得到足夠的高純度iisoA2E進(jìn)行分析。通過(guò)一維及二維核磁共振技術(shù)和質(zhì)譜方法,我們最終確定出了iisoA2E的結(jié)構(gòu)。同A2E及isoA2E的結(jié)構(gòu)的相同點(diǎn)的是iisoA2E也擁有一個(gè)吡啶環(huán)和兩個(gè)多烯側(cè)臂,但其兩個(gè)多烯側(cè)鏈位于吡啶環(huán)的兩個(gè)相鄰碳原子上,這一點(diǎn)與A2E及isoA2E存在較大的差異。iisoA2E是isoA2E的光異構(gòu)體,因而被命名為iisoA2E,但該異構(gòu)體不能直接由A2E的光異構(gòu)化形成。高效液相色譜及質(zhì)譜分析發(fā)現(xiàn)iisoA2E的光氧化活性顯著低于A2E和isoA2E。iisoA2E最大吸收值為430和352 nm,擁有自發(fā)熒光。體外實(shí)驗(yàn)發(fā)現(xiàn)iisoA2E能濃度依賴性抑制RPE細(xì)胞的活力；較高濃度的iisoA2E能破壞細(xì)胞膜的完整性增加LDH的釋放,造成細(xì)胞的嚴(yán)重?fù)p傷。使用磷脂酶D對(duì)A2PE系列產(chǎn)物進(jìn)行處理后,我們檢測(cè)到了除A2E、之外的多種產(chǎn)物,其中就有isoA2E和iisoA2E的存在,這提示isoA2PE及iisoA2PE在體內(nèi)能夠直接合成。綜上,我們?cè)谝暰W(wǎng)膜中發(fā)現(xiàn)一個(gè)新穎的atRAL代謝產(chǎn)物,合成并解析出該產(chǎn)物的結(jié)構(gòu),并研究了其性質(zhì)及體內(nèi)合成途徑,為更好理解類視色素二聚體累積與視網(wǎng)膜變性發(fā)生的關(guān)系及atRAL在體內(nèi)的代謝提供叮實(shí)驗(yàn)支持。
[Abstract]:Age related retinal macular degeneration (age-related macular degeneration, AMD) is a blind eye disease that seriously affects the quality of life in the elderly. A large number of studies have proved that atRAL in the retina is caused by the elimination of metabolic disorders or acute exposure to excessive light exposure in the retina, all trans retinal (all-trans-retinal, atRAL) in the retina. Quantity accumulation may be one of the key causes of macular degeneration in the retina. The molecular mechanism of.AtRAL damage to retina remains to be further clarified. Therefore, this topic chooses retinal pigment epithelium (RPE) cells, which is very important for the maintenance of normal retinal function, and studies the atRAL at the level in vitro. The mechanism of toxic damage to cells and the preliminary exploration of the accumulation of lipofuscin in the fundus of RPE cells for the elimination of atRAL by RPE cells, similar to pigment two polymer (bisretinoids), and there is no unified understanding of the role of these pigments in the pathogenesis of AMD. The accumulation of pigment two polymers has a certain biological activity, so the excess accumulation in the retina may promote retinal degeneration to a certain extent. The composition of the lipofuscin in the retina is extremely complex. The structural analysis of these components, the activity study and the identification of the biocompatibility pathway help us to increase the presence of the retina. Significance and understanding of the metabolism of atRAL in the retina. The final part of this paper is the structural analysis and activity study of a novel atRAL metabolic component in the retina..1. endoplasmic reticulum stress promotes the endogenous atRAL induced by all trans retinol induced retinal pigment epithelial cell apoptosis resulting in RPE cells and sense of RPE cells and feelings. A large number of apoptotic light cells are considered to play a role in the pathogenesis of Stargardt's disease in AMD and adolescent macular degeneration disease. In this chapter, we found that human RPE cell line ARPE-19 can tolerate low concentration of atRAL, but a high concentration of atRAL can cause damage to the cell surface of RPE cells, causing a large number of cells to apoptosis. The step's study found that the treatment of atRAL significantly increased the active oxygen (ROS) in RPE cells and up up the expression of Nrf2, HO-1 and y-GCSh, and caused the oxidative stress reaction of the cells. The subcellular localization found that the ROS in the mitochondria and the endoplasmic reticulum on the mitochondrial and endoplasmic reticulum increased the level of the egg white expression of BiP, and the concentration dependent up regulation of ATF4.CHOP and G. The above results suggest that atRAL induces endoplasmic reticulum stress in RPE cells and activates the PERK-eIF2 alpha -ATF4 pathway. Further studies have found that the endoplasmic reticulum stress inhibitor salubrinal can alleviate the death of RPE cells caused by atRAL, and the antioxidant N- acetyl cysteine (NAC) can effectively inhibit the death and endoplasmic reticulum of RPE cells. Finally, we found that after atRAL treatment, the mitochondrial membrane potential of RPE cells decreased significantly, activated the caspase 3 and PARP. synthesis, and we found that excessive accumulation of atRAL could induce excessive ROS in RPE cells and activate endoplasmic reticulum stress, and endoplasmic reticulum stress aggravated mitochondrial dysfunction of RPE cells, ultimately. Promoting the apoptosis of cell apoptosis.2. retinal pigment epithelial cells to all trans retinol metabolism and the significance of all trans retinol two polymer formation, the excessive accumulation of atRAL in the neural retina will eventually enter the RPE layer, which can cause significant impact on RPE cells. In this chapter, we find RPE cells and atRAL under light condition. After incubation, atRAL can be metabolized to all trans retinol (all-trans-retinol, atROL) and all trans retinaldehyde two polymer (all-trans-retinal dimer, atRAL-dimer), A2E and its isomer, in which the amount of atRAL-dimer is significantly higher than the amount of A2E. Compared with the wild type mice, A2E and its isomer, atRAL-dimer accumulation in RDH8-/-ABCA4 The accumulation and elevation of atRAL-dimer were significantly higher than that of A2E and its isomers. We found that under the same conditions, the inhibitory effect of atRAL-dimer on the activity of RPE cells and the membrane potential of the mitochondria was significantly lower than that of atRAL. The gene Nrf2, HO-1 and gamma GC, which induced the production of reactive oxygen species and oxidative stress, were significantly lower than that of atRAL. The ability of Sh expression is significantly weaker than that in atRAL.. We found that the metabolic atRAL pathway in RPE cells includes the reduction to the atROL pathway and the class of the pigment two polymer pathway represented by atRAL-dimer; both in vivo and in vivo results show that atRAL is easy to accumulate in the cell when atRAL is overdose; and the toxicity is significant after atRAL is converted to atRAL-dimer. Decrease, therefore, the formation of atRAL-dimer is involved in alleviating the toxic damage of atRAL to RPE cells and the discovery of.3. novel retinal lipofuscin composition iisoA2E and its effect on the retinal pigment epithelial cells. It is suggested that the excessive accumulation of lipofuscin in RPE cells may be related to the occurrence of AMD and Stargardt disease. In.RPE lipofuscin there are a large number of autofluorescent properties of the polychrome two polymer, and the role of this component in retinal degeneration needs further clarification. In this chapter, we isolated and identified a novel lipofuscin component iisoA2E. from the retina. We detected that iisoA2E was widely found in mice, cattle, pigs, and mice. In the organic solvent extracts of human eyeballs or RPE/ choroid tissue. The use of atRAL and ethanolamine in vitro can produce iisoA2E, which enables us to obtain sufficient high purity iisoA2E for analysis. We have finally identified the structure of iisoA2E through one-dimensional and two-dimensional NMR and MS methods, with the structure of A2E and isoA2E. The same point is that iisoA2E also has a pyridine ring and two polyene side arms, but its two polyene side chains are located on the two adjacent carbon atoms of the pyridine ring. This point is different from that of A2E and isoA2E..iisoA2E is the photoisomer of isoA2E, so it is named iisoA2E, but the isomer can not be formed directly from the photoisomerization of A2E. High performance liquid chromatography and mass spectrometry analysis showed that the photooxidation activity of iisoA2E was significantly lower than that of A2E and isoA2E.iisoA2E. The maximum absorption value of A2E and isoA2E.iisoA2E was 430 and 352 nm, and there was a spontaneous fluorescence. In vitro, it was found that iisoA2E could inhibit the activity of RPE cells, and the higher concentration of iisoA2E could destroy the integrity of the cell membrane and increase the release of LDH, causing the cell to cause the cell. Severe damage. After the use of phospholipase D to treat A2PE series products, we detected a variety of products except A2E, including the presence of isoA2E and iisoA2E, which suggested that isoA2PE and iisoA2PE can be synthesized directly in the body. To sum up, we found a novel atRAL metabolite in the retina to synthesize and analyze the product. In order to better understand the relationship between the accumulation of retinoid two polymer and the occurrence of retina degeneration and the supporting of atRAL in the metabolism in vivo, the structure of the substance and the synthetic pathway in vivo are studied.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R774.1

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本文編號(hào)：1963737