全反式视黄醛对视网膜色素上皮细胞的毒性作用机制及新颖视网膜脂褐质色素iisoA2E的研究

发布时间：2018-06-01 09:43

本文选题：年龄相关性黄斑变性 + 全反式视黄醛　；参考：《浙江大学》2015年博士论文

【摘要】：年龄相关性视网膜黄斑变性(age-related macular degeneration, AMD)是一种严重影响老年人生活质量的致盲性眼病。大量的研究证明：由于视网膜中全反式视黄醛(all-trans-retinal, atRAL)清除代谢障碍或急性过量光暴露而导致atRAL在视网膜中的过量累积可能是视网膜黄斑变性的关键原因之一。atRAL对视网膜细胞损伤的分子机制有待进一步阐明,因而本课题选择对维持视网膜正常生理功能非常重要的视网膜色素上皮(retinal pigment epithelium, RPE)细胞,在体外水平上研究了atRAL对该细胞的毒性损伤的机制,并初步探索了RPE细胞对atRAL的代谢消除方式。AMD病人眼底累积有大量的脂褐质色素成分——类似色素二聚体(bisretinoids),而人们对这些色素成分在AMD发病中所起到的作用目前尚无统一认识。大量的研究证实类视色素二聚体产物具有一定的生物活性,因而其在视网膜中的过量积聚可能会一定程度促进视网膜变性的进展。视网膜中脂褐质色素的组成极其复杂,对这些成分的结构解析、活性研究与其生物合成通路的确定有助于我们增加对其存在意义及atRAL在视网膜中代谢的认识。因而本论文最后一部分对视网膜中一个新颖的atRAL代谢成分进行了结构解析和活性研究。1.内质网应激促进了全反式视黄醛诱导的视网膜色素上皮细胞凋亡过度累积的内源性atRAL会导致RPE细胞和感光细胞的大量凋亡,这被认为在AMD和青少年型视网膜黄斑变性疾病Stargardt病的发病中起到一定的作用。在本章中,我们发现人RPE细胞系ARPE-19能够耐受低浓度的atRAL,但是较高浓度的atRAL对RPE细胞表现出损伤作用,引起细胞大量凋亡。进一步的研究发现,atRAL的处理使RPE细胞中活性氧(ROS)显著升高,并上调Nrf2, HO-1和y-GCSh的表达,引起细胞氧化应激反应。亚细胞定位发现细胞中的ROS大量定位于线粒体和内质网上。atRAL上调了BiP的蛋白表达水平,并能浓度依赖性上调ATF4.CHOP和GADD34的表达水平。以上结果表明,atRAL引起了RPE细胞内质网应激,激活了PERK-eIF2α-ATF4通路。进一步研究发现,内质网应激抑制剂salubrinal能够一定程度缓解atRAL所致RPE细胞死亡,且抗氧化剂N-乙酰半胱氨酸(NAC)能有效抑制RPE细胞死亡及内质网应激的激活。最后,我们发现经atRAL处理后,RPE细胞的线粒体膜电位明显降低,激活了caspase 3和PARP。综上,我们发现过度累积的atRAL可诱导RPE细胞内产生过量的ROS并激活细胞内质网应激,内质网应激加重了RPE细胞的线粒体功能障碍,最终促进了细胞的凋亡。2.视网膜色素上皮细胞对全反式视黄醛的代谢及全反式视黄醛二聚体形成意义的探索在神经视网膜层过度积聚的atRAL最终会进入RPE层,从而能够对RPE细胞造成显著影响。在本章中,我们发现避光条件下RPE细胞和atRAL孵育后,atRAL可以被代谢为全反式视黄醇(all-trans-retinol,atROL)和全反式视黄醛二聚体(all-trans-retinal dimer,atRAL-dimer)、A2E及其异构体等产物,其中atRAL-dimer的量显著高于A2E的量。同野生型小鼠相比,A2E及其异构体、atRAL-dimer的累积量在RDH8-/-ABCA4-/-小鼠眼球中显著升高,但atRAL-dimer的累积量及升高幅度均显著高于A2E及其异构体。我们发现同等条件下atRAL-dimer对RPE细胞活力、细胞线粒体的膜电位的抑制作用显著低于atRAL,其诱导细胞活性氧产生及氧化应激的相关基因Nrf2,HO-1和γGCSh表达的能力显著弱于atRAL.综上,我们发现在RPE细胞中的代谢atRAL途径包括还原为atROL通路和以atRAL-dimer为代表的类视色素二聚体生成通路；体内外的结果均表明,atRAL过量时易于在细胞内累积atRAL-dimer;atRAL转变为atRAL-dimer后,毒性显著降低,因此atRAL-dimer的生成参与缓解atRAL对RPE细胞的毒性损伤。3.新颖视网膜脂褐质色素成分iisoA2E的发现及其对视网膜色素上皮细胞的影响大量的研究表明,在RPE细胞中过度累积的脂褐质成分与AMD及Stargardt病的发生可能存在一定的关联。RPE脂褐质中存在大量的具有自发荧光特性的类视色素二聚体成分,该类成分在视网膜变性中的作用尚需进一步阐明。在本章,我们从视网膜中分离鉴定出一个新颖的脂褐质色素成分iisoA2E。我们检测到iisoA2E广泛存在于小鼠、牛、猪及人的眼球或RPE/脉络膜组织有机溶剂提取物中。在体外利用atRAL和乙醇胺反应能够制得iisoA2E,使得我们能够得到足够的高纯度iisoA2E进行分析。通过一维及二维核磁共振技术和质谱方法,我们最终确定出了iisoA2E的结构。同A2E及isoA2E的结构的相同点的是iisoA2E也拥有一个吡啶环和两个多烯侧臂,但其两个多烯侧链位于吡啶环的两个相邻碳原子上,这一点与A2E及isoA2E存在较大的差异。iisoA2E是isoA2E的光异构体,因而被命名为iisoA2E,但该异构体不能直接由A2E的光异构化形成。高效液相色谱及质谱分析发现iisoA2E的光氧化活性显著低于A2E和isoA2E。iisoA2E最大吸收值为430和352 nm,拥有自发荧光。体外实验发现iisoA2E能浓度依赖性抑制RPE细胞的活力；较高浓度的iisoA2E能破坏细胞膜的完整性增加LDH的释放,造成细胞的严重损伤。使用磷脂酶D对A2PE系列产物进行处理后,我们检测到了除A2E、之外的多种产物,其中就有isoA2E和iisoA2E的存在,这提示isoA2PE及iisoA2PE在体内能够直接合成。综上,我们在视网膜中发现一个新颖的atRAL代谢产物,合成并解析出该产物的结构,并研究了其性质及体内合成途径,为更好理解类视色素二聚体累积与视网膜变性发生的关系及atRAL在体内的代谢提供叮实验支持。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R774.1

【相似文献】