视网膜维生素A循环的研究进展

维生素A循环是人类视觉形成的基础,它为光反应提供了生色团,并从一定意义上维持了人体内维生素A类物质的浓度.本文综述了维生素A循环的相关研究,阐明了各步代谢过程以及相关酶类的作用机制,并对感光细胞内全反式视黄醛转变为全反式视黄醇、色素上皮细胞内11-顺-视黄醇转变为11-顺-视黄醛以及两者之间的转运和异构化作了总结.     

脂褐质荧光基团N-亚视黄基-N-视黄基乙醇胺在人视网膜色素上皮细胞蓝光损伤中的作用研究

目的探讨成人视网膜色素上皮（hRPE）细胞对脂褐质荧光基团N-亚视黄基-N-视黄基乙醇胺（A2E）的摄取及A2E在hRPE细胞蓝光损伤中的作用。方法全反式视黄醛和乙醇胺混合,体外合成A2E。体外培养hRPE细胞。将A2E加入hRPE细胞培养液中,使A2E的浓度分别为25、50、100μmoL／L。观察hRPE细胞对A2E的摄取。用蓝光（450nm）照射hRPE细胞20min,光照强度70mW／mm^2,CCK-8检测细胞活力,Hoechst 33342荧光素染色、流式细胞仪检测细胞凋亡情况。结果全反式视黄醛（100．0mg）和乙醇胺（9．5mg）混合后生成53．8mg的A2E。吞噬A2E的hRPE细胞中能够检测到自发荧光,主要分布在细胞核周围。蓝光照射后,含有A2E的hRPE细胞活力下降,下降程度随A2E浓度增加而增强。不含A2E的hRPE细胞,经蓝光照射后,细胞活力未见明显下降。蓝光照射含有A2E的hRPE细胞,细胞核中出现浓染的碎块状荧光,流式细胞仪检测可见凋亡细胞特有的亚二倍体峰,加入25μmoL／L的A2E,光照后12、24、36及48h,细胞凋亡率分别为（12．11±2．32）％、（31．21±3．72）％、（64．23±3．53）％及（58．71±3．48）％。仅接受光照不加入A2E、不接受光照（加入或不加入A2E）的hRPE细胞凋亡率在光照后各时间点的平均值均小于5％。结论在hRPE细胞蓝光损伤的过程中,单独的光照射不会造成明显的细胞损伤或凋亡,A2E的存在是蓝光损伤的因素之一。提示在老年人中,含有较多脂褐质的hRPE细胞容易受到蓝光损伤。（中华眼科杂志．2007,43：1017-1021）     

全反式维生素A酸对抑制视网膜母细胞瘤株增殖的实验研究

全反式维生素A酸(all-trans retinoic acid，AT-RA)能抑制肿瘤细胞的增殖而达到治疗肿瘤的目的。我们采用AT-RA对视网膜母细胞瘤(Retinoblastoma，RB)株进行增殖抑制的实验研究，以期探索肿瘤治疗的新途径。     

眼底白点状视网膜变性和锥细胞营养障碍的年轻单卵双胞胎姐妹

目的：描述一对年轻的单卵双胞胎姐妹，眼底呈现白点状视网膜变性[一种常染色体隐性遗传病，由11-顺视黄醇脱氢酶基因（RDI-15）突变而引起静止性夜盲]合并锥细胞营养障碍，此病之前在老年男性中曾有报道。     

视黄醛在实验性近视中的作用

近视是一种发育性疾病，近视眼球巩膜的主动扩张是其伸长的重要机制，而视黄醛是眼球发育中重要的信号转导分子，其在脊椎动物的眼球发育中具有多种不同的重要作用。视黄醛可能是调节实验性近视眼球伸长的信使分子近年来有关视黄醛与实验性近视发生，发展的关系的研究取得一定进展，本研究综述了视黄醛及其核受体，实验性近视眼球的视网膜，脉络膜，巩膜的视黄醛改变以及视黄醛作为传递从视网膜到巩膜的眼球伸长信号的信使分子的研究进展情况。     

全反式维甲酸在眼表疾病中应用的研究进展

全反式维甲酸(all-trans retinoid acid,ATRA)是维生素A的重要中间代谢物,其在调节眼部组织多种细胞的增生、分化以及维持上皮组织的正常角化等过程中均发挥重要作用.ATRA在角结膜瘢痕性病变如Stevens-Johnson综合征以及角膜损伤性疾病中可能具有独特的疗效,同时ATRA在抑制角膜移植术后的免疫排斥反应和治疗局部角膜缘干细胞缺乏症也具有良好的应用前景.     

不同波长的蓝光对人视网膜色素上皮细胞的影响

目的：探讨不同波长的蓝光对人视网膜色素上皮细胞(RPE)的影响。

方法：将体外培养的ARPE-19细胞随机分为对照组、447nm蓝光组、456nm蓝光组、468nm蓝光组,对照组细胞于常规条件下培养,蓝光组细胞使用光强为200Lx的OLED蓝光背光源照射72h,利用细胞活/死染色实验、CCK-8实验、Real-time PCR等方法比较不同波长的蓝光对细胞形态、细胞活性、增殖能力及视循环功能指标和炎症指标mRNA表达的影响。

结果：蓝光照射后,ARPE-19细胞的形态发生变化,细胞融合减少。蓝光波长越短,对细胞增殖抑制作用越明显,细胞内增殖标志物Ki-67 mRNA表达越少,视循环功能指标卵磷脂视黄醇酰基转移酶(LRAT)、视黄醛结合蛋白(CRALBP)、视黄醛脱氢酶(RDH)、光受体视黄醇类结合蛋白(IRBP)mRNA表达下调越明显,细胞内炎症因子单核细胞趋化因子(MCP-1)、白介素-6(IL-6)mRNA表达水平上调越明显。

结论：不同波长蓝光对RPE细胞均有损害作用,且蓝光波长越短,其损害作用越大。     

视黄酸在实验性近视发生中作用的研究进展

近视是一种常见的导致视力障碍的眼科疾病,其患病率逐年升高,而目前临床上尚无有效的根治手段,原因在于近视的发病机制尚不明确.目前与近视发生相关的因子很多,如多巴胺、视黄酸、胰高血糖素及ZENK(Zif269、EGR-1、NGFI-A或Krox-24)等.视黄酸是维生素A的衍生物,是视网膜光感受器细胞中视蛋白结合物视黄醛的最终代谢产物.许多研究表明视网膜与脉络膜中的视黄酸在近视发生中起到重要作用,可以调节近视相关的多种因子的作用,如转化生长因子-β、糖胺聚糖、基质金属蛋白酶(MMP)-2、金属蛋白酶组织抑制剂(TIMP)-2等.本文基于眼内视黄酸的产生、代谢及作用,就视黄酸在形觉剥夺、光学离焦及色光诱导性近视等3种实验性近视发生中的作用做一综述,以期对今后的近视机制研究带来一些启发.     

维生素A在眼表面作用的研究进展

维生素A对于正常角结膜上皮的生长和分化十分重要，本综述了维生素A的作用机制、与角结膜上皮细胞分化的关系以及维生素A制剂在干眼症中的应用。     

与白色点状视网膜炎相关细胞Retinaldehyde黏连蛋白基因（RLBP1）的突变 异型结合体中家族间基因多相性和眼底改变的证据

目的：在来自3个家族中的5例患者中评价与白色斑点性视网膜炎(RPA)的分子遗传学缺陷。方法：我们检查了患有RPA的3例先证者和2例临床受累者。临床检查包括最佳视力检查，视野检查，视网膜电图检查，双眼眼底检查和眼底照相。对白细胞的DNA进行分析，在基因编码细胞retinaldehyde-黏连蛋白1(RLBP1)，11-cis-视黄醇脱氢酶中(RDH5)，(RBP3)，光感受器之间的类维生素黏连蛋白(RBP3)和光感受器全反视黄醇脱氢醇(RDH8)的外显子中找到突变。不是所有的患者都进行了全部基因的突变评估。外显子通过单链构象多态性分析或直接基因测序被分别的倍增和筛选，以寻找突变。     

Interphotoreceptor retinoid-binding protein contains three retinoid binding sites

Interphotoreceptor retinoid binding protein (IRBP), the major soluble protein component of the interphotoreceptor matrix, is believed to participate in the visual cycle by transporting retinoids between retinal pigment epithelium and photoreceptor cells in the eye. IRBP can associate with several chemical and isomeric forms of retinoids but displays the highest affinity towards the retinoids that are important in the visual cycle, 11-cis-retinal and all-trans-retinol. It was previously reported that IRBP can associate with 2 mol of all-trans-retinol or 2 mol of 11-cis-retinal per mol of protein. One of the retinoid binding sites, termed 'site 1', was found to display a broad ligand selectivity and to bind either all-trans-retinol or 11-cis-retinal with similar affinities. Here, the retinoid-binding properties of IRBP were further examined. The data demonstrate that IRBP contains three distinct retinoid binding sites. The promiscuous 'site 1', and two additional sites with a stricter selectivity. One of the latter sites appears to be selective towards all-trans-retinol, while the other is specific for 11-cis-retinal.     

Confronting complexity: the interlink of phototransduction and retinoid metabolism in the vertebrate retina

Absorption of light by rhodopsin or cone pigments in photoreceptors triggers photoisomerization of their universal chromophore, 11-cis-retinal, to all-trans-retinal. This photoreaction is the initial step in phototransduction that ultimately leads to the sensation of vision. Currently, a great deal of effort is directed toward elucidating mechanisms that return photoreceptors to the dark-adapted state, and processes that restore rhodopsin and counterbalance the bleaching of rhodopsin. Most notably, enzymatic isomerization of all-trans-retinal to 11-cis-retinal, called the visual cycle (or more properly the retinoid cycle), is required for regeneration of these visual pigments. Regeneration begins in rods and cones when all-trans-retinal is reduced to all-trans-retinol. The process continues in adjacent retinal pigment epithelial cells (RPE), where a complex set of reactions converts all-trans-retinol to 11-cis-retinal. Although remarkable progress has been made over the past decade in understanding the phototransduction cascade, our understanding of the retinoid cycle remains rudimentary. The aim of this review is to summarize recent developments in our current understanding of the retinoid cycle at the molecular level, and to examine the relevance of these reactions to phototransduction.     

Analysis of the visual cycle in cellular retinol-binding protein type I (CRBPI) knockout mice

PURPOSE: To determine whether the visual cycle is affected in mice without a functional gene for cellular retinol-binding protein type I (CRBPI(-/-) mice). METHODS: Visual-cycle retinoids and rhodopsin levels were analyzed in eyes of dark adapted (DA) CRBPI(-/-) and wild-type (wt) mice before and during recovery from a flash. The rate of dark adaptation was analyzed using electroretinography (ERG). RESULTS: all-trans-retinyl esters were reduced to approximately 33% of wt levels in DA CRBPI(-/-) mice. Recovery from a flash in wt mice produced transient accumulations of all-trans-retinal and all-trans-retinyl ester, as the pulse of retinoid produced by the flash traversed the visual cycle. In CRBPI(-/-) mice, all-trans-retinal accumulated transiently, as in wt mice. However, all-trans-retinol also accumulated transiently in the neural retina, and the transient increase in all-trans-retinyl ester of the wt was reduced. Rates of 11-cis-retinal and rhodopsin formation were comparable in wt and CRBPI(-/-) mice. Dark adaptation was delayed by a factor of approximately two. CONCLUSIONS: The accumulation of all-trans-retinol in neural retina, in the absence of CRBPI and the reduced amount of retinyl esters in the RPE suggest that the binding protein participates in a process that drives diffusion of all-trans-retinol from photoreceptor cells to RPE, perhaps by delivering vitamin A to lecithin-retinol acyltransferase (LRAT) for esterification. Because the perturbation occurred upstream of a slow step of the visual cycle, there was no major impairment of the rate of visual pigment regeneration.     

Acyl CoA:retinol acyltransferase (ARAT) activity is present in bovine retinal pigment epithelium

Visual perception is mediated by a family of G protein-coupled receptors called the opsins. The light-absorbing chromophore in most opsins is 11-cis-retinaldehyde, which is isomerized to all-trans-retinaldehyde upon absorption of a photon. Restoration of light sensitivity to the photobleached opsin requires chemical re-isomerization of the chromophore. This is carried out by an enzymatic pathway called the visual cycle in retinal pigment epithelial cells. The isomerase in this pathway uses fatty-acyl esters of all-trans-retinol as substrate. A retinyl-ester synthase that produces these esters, called lecithin:retinol acyltransferase (LRAT), has been extensively characterized. Based on prior biochemical studies and the phenotype in lrat(-/-) knockout mice, it has been assumed that LRAT is the sole or dominant retinyl-ester synthase in the retinal pigment epithelium. Here we demonstrate the presence of a second ester synthase activity in these cells called acyl CoA:retinol acyltransferase (ARAT). We show that this activity uses palmitoyl coenzyme A as an acyl donor, unlike LRAT which uses phosphatidylcholine. Similar to LRAT, ARAT esterifies both all-trans-retinol and 11-cis-retinol. LRAT and ARAT are both potently inhibited by the retinyl-ester analog, all-trans-retinylbromoacetate, but only ARAT is inhibited by progesterone. Unexpectedly, the maximum turnover rate (V(max)) of ARAT was similar to that of LRAT. However, the Michaelis constant (K(M)) of ARAT was 10-fold higher than the K(M) of LRAT for all-trans-retinol. These observations suggest that ARAT may complement LRAT to provide additional retinyl-ester synthase activity under conditions of high all-trans-retinol. These conditions occur in the retina following exposure to bright light.     

Isolation and characterization of a retinal-binding protein from the squid retina

A retinal-binding protein (RALBP) was isolated from the squid retina, and purified by anion-exchange and size-exclusion chromatography. The molecular weight was determined to be 51,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration. The purified sample showed absorption maxima at about 330 and 400 nm in addition to a protein band, indicating the occurrence of retinol and retinal, respectively. The relative heights of these two peaks varied from preparation to preparation, depending on retinoid ligands. Irradiation of RALBP caused no marked change in absorption, but the amount of 11-cis-retinal decreased to form a photosteady state mixture with all-trans- and 13-cis-retinals. RALBP was fairly stable even in the presence of hydroxylamine (100 mM), but was affected by sodium borohydride (30 mM) or borane dimethylamine (400 mM), with the retinal reduced to retinol. When incubated with metaretinochrome-carrying membranes in the dark, RALBP specifically took up 11-cis-retinal and lost all-trans-retinol. Upon further incubation of this RALBP with opsin-containing membranes, rhodopsin was progressively formed in the dark. Squid RALBP may act as a shuttle in transferring the 11-cis-retinal from metaretinochrome to opsin in the visual cells.     

Uptake and isomerization of all-trans retinol by isolated bovine retinal pigment epithelial cells: further clues to the visual cycle

The site and substrate for all-trans to 11-cis isomerization in the visual cycle have remained obscure for several decades. Only recently studies on a subcellular level have begun to shed some light on these phenomena. We have addressed this system on a cellular level by utilizing intact isolated bovine retinal pigment epithelial cells, maintained during short-term incubation in vitro. Supplementation with labeled all-trans retinol incorporated in a lipid vesicle carrier, in a range of 1-6 nmol per 10(6) cells, resulted in a rapid uptake of retinol. The majority of the internalized retinol was processed prior to mixing with endogenous retinoid pools and most of it was converted into all-trans retinylester. Up to 10% of the incorporated label was isomerized yielding 11-cis retinol, 11-cis retinaldehyde and 11-cis retinylester. The kinetics of the 11-cis retinoid formation indicated that 11-cis retinol is the first isomerization product. The level of 11-cis retinol apparently 'triggered' further processing into other 11-cis retinoids. An updated model with discussion topics is presented for the retinoid pathway relevant to the visual cycle.     

Properties and immunocytochemical localization of three retinoid-binding proteins from bovine retina

Cellular retinal-binding protein (CRALBP) complexed with 11-cis-retinal has several properties characteristic of a visual pigment. Interaction of the protein and retinoid results in a bathochromic shift in the absorption spectrum of the chromophore from 380 to 425 nm, accompanied by a decrease in the extinction coefficient (25,000-15,000 M-1 cm-1). Illumination of the complex results in the progressive loss of absorbance at 425 nm and an increase at 375 nm, consistent with the production of a geometrical isomer of retinal that lacks affinity for the binding protein. Analysis by HPLC of the retinoids after illumination reveals that the basis of the spectral transition is a photoisomerization of 11-cis-retinal to all-trans-retinal. Only small amounts (less than 10%) of 13-cis-retinal are produced during the photoisomerization, showing the stereospecificity of the process. Although CRALBP has the spectral characteristics of a blue-sensitive visual pigment, there is no evidence that this is related to its function. This protein may serve as a model for the interactions of 11-cis-retinal and protein. Eleven-cis-retinol bound to CRALBP is a better substrate for esterification by microsomes from retinal pigment epithelium (RPE) than all-trans-retinol bound to cellular retinol-binding protein (CRBP). The product of the reaction, retinyl ester, does not remain bound to either binding protein but becomes associated with the microsomal fraction. Esterification is the first described process, occurring in the dark, by which retinoids can be removed from CRBP and CRALBP. Antibodies to bovine CRBP have been produced in rabbits following injection of the performic acid-oxidized protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake, processing and release of retinoids by cultured human retinal pigment epithelium

Upon absorption of a photon, the 11-cis retinaldehyde chromophore of rhodopsin is isomerized and reduced to all-trans retinol (vitamin A) in the photoreceptor outer segments, whereupon it leaves the photoreceptors, and moves to the retinal pigment epithelium (RPE). To clarify the function of the RPE in the regeneration of 11-cis retinaldehyde, we delivered all-trans retinol to monolayer cultures of human RPE. During delivery the retinol was associated with its putative natural carrier, interphotoreceptor retinoid binding protein (IRBP). IRBP has been proposed as a carrier protein involved in the exchange of retinoids between the photoreceptors and the retinal pigment epithelium. The retinoid composition of RPE cells and culture medium was analyzed by HPLC following several incubation periods. The RPE monolayer was found to process all-trans retinol into two distinct end-products: all-trans retinyl palmitate, which remained within the RPE monolayer: and 11-cis retinaldehyde which was released into the culture medium. These results demonstrate retinoid isomerase, retinol oxidoreductase and retinyl ester synthetase activity in human RPE cells cultured under the appropriate conditions. They show that IRBP can serve as a carrier of retinol through an aqueous medium to the RPE, and they illustrate that the visual cycle can be studied in vitro.