褪黑素对高糖刺激人视网膜色素上皮细胞诱导型一氧化氮合酶表达的影响

目的研究褪黑素对高糖刺激体外培养的人视网膜色素上皮（retinal pigment epithelial,RPE）细胞诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）表达的影响。方法培养人RPE细胞,分为对照组、甘露醇组、高糖组和高糖＋褪黑素组,作用48h后,光镜观察细胞形态;MTT法检测细胞活性;免疫荧光染色法和Western blot检测RPE细胞中iNOS的蛋白表达。结果MTT检测结果表明高糖可以抑制RPE细胞增生,加入褪黑素后,抑制作用减弱;免疫荧光染色法和Western blot检测结果表明,与对照组相比,高糖组iNOS蛋白表达明显增高,加入褪黑素后,iNOS表达被显著抑制。结论高糖可以抑制RPE细胞增生,诱导RPE细胞iNOS的表达上调,而褪黑素可减轻细胞损伤。     

神经生长因子对激光致视网膜损伤后bFGF蛋白表达的影响

目的观察神经生长因子（NGF）对激光致视网膜损伤后内源性碱性成纤维细胞生长因子（bFGF）蛋白表达的影响，以探讨激光致视网膜损伤的分子病理机制。方法Wistar二级大鼠，分为3组，即损伤对照组、生理盐水对照组、NGF组，分别于倍频Nd：YAG激光损伤后，玻璃体内注射NGF，用免疫组织化学、Western blot方法、苏木精-伊红染色及视觉电位仪测量视网膜电图（ERG）b波幅值，观察视网膜bFGF蛋白表达量的变化及对视网膜组织结构、视功能的影响。结果激光损伤视网膜后玻璃体内NGF组3dbFGF蛋白的表达明显低于损伤对照组（P〈0．01）；NGF组28d感光细胞结构的恢复较损伤对照组差，NGF组的b波幅值在各时间点与对照组比较差异均无统计学意义（P〉0．05）。结论外源性NGF减少了激光视网膜损伤后bFGF产物，不利于感光细胞结构和功能的修复。     

实验性视网膜脱离模型中视网膜色素上皮细胞黏附分子的表达

目的观察大鼠视网膜脱离及复位状态下视网膜色素上皮（RPE）细胞黏附分子的表达变化，探讨其与增生性玻璃体视网膜病变（PVR）发生的关系。方法通过视网膜下注射透明质酸钠的方法制造视网膜脱离的动物模型。在不同的时间点摘除眼球，制作冰冻切片，进行免疫组织化学及免疫荧光染色，比较RPE细胞上几种黏附分子，包括神经钙粘素、上皮钙粘素、整合素α5、整合素β1，以及纤维连接蛋白（FN）在正常视网膜、脱离的视网膜、复位的视网膜中的表达情况。结果几种黏附分子在脱离区的RPE细胞上的表达均明显高于正常视网膜以及脱离后复位的视网膜。结论视网膜脱离会导致RPE细胞上几种重要的黏附分子的表达增加，视网膜复位可逆转此种变化。视网膜脱离可能是PVR发生的始动因素。     

光损伤鼠视网膜片培养上清液诱导 MSCs 分化为视网膜样细胞的研究

目的：应用大鼠视网膜片光损伤后的培养上清液,在体外诱导大鼠骨髓间充质干细胞（ mesenchymal stem cells , MSCs）成为视网膜样细胞的可能性。 方法：贴壁筛选法分离、培养大鼠MSCs ,流式细胞仪对其细胞纯度鉴定。取材大鼠视网膜神经上皮层作为视网膜片,常规石蜡切片HE染色鉴定各层组织完整性。电镜观察大鼠视网膜片光损伤程度。制备3种诱导分化大鼠MSCs的条件培养液。3种条件培养液均培养诱导至第3代大鼠MSCs 7～8d,用RT-PCR检测视紫红质（Rhodopsin）、神经元特异性烯醇化酶（neuron-specific enolase,NSE）、胶质纤维酸性蛋白（ glial fibrillary acidic protein ,GFAP）等视网膜细胞标志物在诱导后细胞中的表达情况。 结果：HE染色显示大鼠视网膜片取材结构完整,电镜显示大鼠视网膜片光损伤后结构损伤严重。 RT-PCR鉴定：条件培养液诱导大鼠MSCs 7～8d,条件培养液Ⅰ组Rhodopsin （0．3915±0．00644）、NSE （0．2019±0．00682）、GFAP （0.1972±0．00211）,条件培养液Ⅱ组Rhodopsin（0．0983±0．00319）、NSE （0．1048±0．00323）、GFAP （0．1040±0.00254）,条件培养液Ⅲ组Rhodopsin（0．0044±0．00126）、NSE（0．0498±0．00149）、GFAP（0．0467±0．00333）,组间差异有统计学意义。 结论：光损伤大鼠视网膜片培养上清液可诱导大鼠MSCs分化为视网膜样细胞,为干细胞治疗视网膜变性疾病提供新思路。     

大鼠视神经损伤后一氧化氮合酶的变化及牛磺酸的影响

目的研究大鼠视神经挫伤后诱导型一氧化氮合酶（iNOS）的变化及牛磺酸的影响。方法通过建立外伤性视神经损伤的动物模型,随机分为正常对照组、损伤组（视神经钳夹＋蒸馏水组）及牛磺酸组（视神经钳夹＋牛磺酸组）,于损伤后3d、7d、14d和28d以免疫组化染色法测定视神经iNOS的活性。结果在损伤后3d、7d、14d及28d,损伤组及牛磺酸组视神经组织iNOS表达较正常对照组升高（F=256．43,213．62,188．76,231．78,P〈0．05）;牛磺酸组较损伤组iNOS表达降低（F=256．43,213．62,188．76,231．78,P〈0．05）。结论视神经损伤诱导视神经iNOS的表达。牛磺酸可能通过对iNOS的抑制在大鼠视神经损伤中起到视神经保护作用。     

一氧化氮及其合酶在糖尿病视网膜损伤中的作用

目的进一步探讨一氧化氮（NO）及其合酶在糖尿病视网膜氧化损伤中的作用。方法链脲佐菌素制备大鼠糖尿病模型,注射生理盐水作为对照,分别取正常对照及模型制备成功后2周及20周大鼠视网膜标本进行以下实验：（1）利用免疫组化及图像处理技术,分析硝基酪氨酸在视网膜中的分布及含量;（2）利用免疫组化及RT—PCR技术,测定iNOS及nNOS的蛋白及mRNA表达。结果糖尿病大鼠视网膜内的3-硝基酪氨酸（3-NT）在糖尿病2周大鼠的表达升高,但阳性细胞只分布于神经节细胞层,提示糖尿病大鼠的视网膜损伤首先发生在神经节细胞层。糖尿病20周的大鼠3-NT表达量明显增高,并且阳性细胞遍布视网膜全层,显示随糖尿病病程进展视网膜氧化损伤进行性加重,NO产生增多,和对照相比,糖尿病2周大鼠的iNOS表达增加,nNOS表达下降,糖尿病20周大鼠的iNOS进一步增加,而nNOS几乎消失,提示糖尿病大鼠视网膜内NO产生增多和nNOS表达下降与iNOS表达升高有关。结论糖尿病对视网膜组织的损害首先是发生在神经节细胞,进而外层视网膜功能受损,糖尿病早期视网膜组织的损伤是以神经节细胞层为主;糖尿病大鼠模型中,视网膜损伤和NO的升高关系密切,而NO含量升高是nNOS表达下降和iNOS表达升高的结果。（中华眼科杂志,2005,41：837-841）     

银杏叶提取物对大鼠视网膜光损伤后感光细胞的保护作用

目的:探讨银杏叶提取物（EGb 761）对视网膜光损伤后感光细胞 的保护作用。 方法:72只Sprague-Dawley(SD)大鼠随机分为正常对照组 、模型组、模型+生理盐水组和模型+ EGb 761组，每组 各18只大鼠。模型组、模型+生理盐水组和模型+EGb 761组暗适应24 h后持续光照6 h，光照强度为（2740±120）lx ，建立光损伤模型。模型+EGb 761组和模型+生理盐水组分别于光照前7 d每日腹腔注射0.35 % EGb 761（100 mg/kg）和相应体积的生理盐水，光照后继续给药14 d；于光照后4 d对 各组进行视网膜原位凋亡细胞检测，并于光照后7、14 d作组织病理学检查并计数视盘 上、下方视网膜外核层（ONL）感光细胞层数。 结果:光照后4 d，除 正常对照组外，其余 各组ONL均出现凋亡感光细胞，但模型+ EGb 761组ONL凋亡感光细胞数明显少于模型组和模 型+生理盐水组。光照后7 d，模型组和模型+生理盐水组感光细胞核层数均为3~4层，模型+ EGb 761组为7~8层；模型+ EGb 761组平均感光细胞核层数（6.92 ± 0.82）少于正常对照 组（8.40±0.95）（t=-1.416，P＜0.05），但显著多于模型组（5.96±1.36）和 模型+生理盐水组（5.90±1.40）（t=1.024，1.084；P＜0 .05）。光照后14 d，模型 组和模型+生理盐水组感光细胞核为0~1层，而模型+ EGb 761组仍存有3~4层；模型+ EGb 76 1组平均感光细胞核层数（5.52±1.06）仍显著多于模型组（3.44±2.15）和模型+ 生理 盐水组（3.37±1.91）（t=2.082，2.146；P＜0.05）。 结论:EGb 761能部分抑制视网膜光损伤后感光细胞凋亡，对感光细胞具 有一定的保护作用。     

视网膜重度激光损伤和修复的组织学观察

目的观察重度激光光凝对视网膜脉络膜的损伤及其组织的修复。方法家兔用1％阿托品液扩瞳后，采用Q-开关红宝石激光多脉冲辐照，光照当日及以后的不同时期作检眼镜观察，共同时期处死家兔，取出眼球作光镜和电镜观察、实验同期3个月。结果（1）光凝的损伤：光照当日见：光照处视网膜破裂消失，Bruch膜及大部分的脉络膜组织亦破坏消失，光照周围视网膜水肿，高起弯曲，内颗粒层结构破坏明显，并见视网膜下积液。光照周围的视网膜、视网膜下及玻璃体出血。（2）组织的修复：修复过程目光换后第3天即可见．此时光照周围的脉络膜成纤维细胞增生并渐增生活跃伸向光照区以修复视网膜脉络膜破坏消失处。光照周围视网膜的损伤由胶质细胞增生以修复。光照处未见Bruch膜和色素上皮（RPE）的再生与修复。结论重度红宝石激光能造成视网膜脉络膜较大的损伤，使光照处视网膜脉络膜组织破坏消失，其周围的视网膜亦受环境影响，出现水肿和神经上皮层的破坏。光照处的组织损伤主要由纤维组织和胶质细胞增生以修复，光照处被破坏的BM和RPE未见再生与修复。     

岩茶提取物对大鼠视网膜光损伤的保护作用

目的　探讨岩茶提取物对大鼠视网膜光损伤的保护作用。方法　４０只ＳＤ大鼠随机分为４组:正常对照组、光损伤组、高剂量组（０．２０ｇ?Ｌ－１）和低剂量组（０．０５ｇ?Ｌ－１）。除正常对照组外,其余三组置于光照强度为（４０００±２００）Ｌｕｘ的光照箱内照射１２ｈ。高、低剂量组光照前３ｄ给予岩茶提取物尾静脉注射,每天一次,光照结束后继续给药７ｄ后处死。光镜下观察４组视网膜病理变化,检测视网膜组织外核层厚度、超氧化物歧化酶（ｓｕｐｅｒｏｘｉｄｅｄｉｓｍｕｔａｓｅ,ＳＯＤ）活力及丙二醛（ｍａｌｏｎａｌｄｅｈｙｄｅ,ＭＤＡ）含量。结果　光损伤组光镜下视网膜各层结构疏松,外核层厚度变薄,见大量空泡细胞;感光细胞内外节排列紊乱,染色不均。高、低剂量组损伤均较光损伤组轻,其中高剂量组损伤更轻。与正常对照组（４１．０６±１．０１）μｍ比较,其余三组视网膜外核层厚度变薄（均为Ｐ＜０．０５）,高、低剂量组较光损伤组（１５．１０±１．９２）μｍ厚,其中高剂量组（２５．７７±１．０８）μｍ较低剂量组（１９．２４±０．５５）μｍ厚（Ｐ＜０．０５）。与正常对照组相比,其余三组视网膜组织ＳＯＤ活力下降（Ｐ＜０．０５）,ＭＤＡ含量升高（Ｐ＜０．０５）;与光损伤组比较,高、低剂量组视网膜ＳＯＤ活力升高（Ｐ＜０．０５）,ＭＤＡ含量下降（Ｐ＜０．０５）,高剂量组表现更明显（Ｐ＜０．０５）。结论　岩茶提取物对大鼠视网膜光损伤有一定的保护作用,且具有剂量依赖性。     

结缔组织生长因子在体外人视网膜色素上皮 细胞损伤模型中的表达和促移行作用

目的观察结缔组织生长因子（CTGF）在视网膜色素上皮（RPE）细胞损伤模型中的表达及其对RPE细胞移行的影响。方法利用体外培养的单层近融合期人RPE细胞，采用棉签和角膜移植用环钻做圆形细胞刮伤区，建立体外RPE细胞损伤模型。链霉亲和素-生物素复合物（SABC）免疫组织化学和原位杂交（ISH）方法检测RPE细胞受创后不同时间CTGF的表达及其转录水平mRNA的变化。并计数进入缺损区的RPE细胞数，定量观察CTGF对RPE细胞移行的影响以及地塞米松对CTGF这一作用的影响。结果免疫组织化学和原位杂交结果显示,创伤后6 h，创伤边缘RPE细胞表达CTGF呈弱阳性反应; 随着创伤后时间延长,阳性信号逐渐增强；伤后24、48 h创伤边缘移行的RPE细胞呈CTGF强阳性表达。重组人CTGF因子（rhCTGF）呈剂量依赖性刺激RPE细胞移行，地塞米松对CTGF诱导的RPE细胞移行作用有显著抑制作用。结论CTGF参与了RPE细胞创伤修复过程，这在增生性玻璃体视网膜病变等眼内增生性疾病中可能有重要意义。(中华眼底病杂志,2005,21:306-309)     

Retinal pigment epithelial cell transplantation in RCS rats: normal metabolism in rescued photoreceptors.

Photoreceptor cells in Royal College of Surgeons (RCS) rats with inherited retinal dystrophy can be rescued by the transplantation of normal, wild-type retinal pigment epithelial (RPE) cells, if done before photoreceptor cell death. In the present study, we have examined several metabolic features of rescued photoreceptors and transplanted RPE cells at 2.3-3.3 months after transplantation. Rescued photoreceptors with a structurally normal RPE interface showed a rod outer segment renewal rate similar to that of normal control rats of 2.2 microns day-1, as measured in autoradiograms. Rod outer segment disc shedding had values indistinguishable from those in normal controls, as measured by the number of phagosomes in the transplanted RPE cells both during the burst of disc shedding soon after the onset of light in the morning and during the middle of the light cycle when disc shedding is low. The interphotoreceptor matrix, which is synthesized by both photoreceptors and the RPE, was distributed normally in the regions where normal-appearing photoreceptors were present underlying normal, transplanted RPE cells. Thus, the rescued photoreceptors show normal metabolic rates and normal interactions with the RPE in each of the parameters examined. These findings, combined with the previous demonstration of opsin and Na+,K(+)-ATPase expression by the rescued photoreceptors, support our interpretation that the surviving, normal-appearing photoreceptors may function normally. Moreover, transplantation of normal RPE cells reversed pathological changes in the photoreceptors that had already occurred by the time of transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)     

鼠视网膜光化学损伤中的主要组织化学改变

目的：观察大鼠视网膜光化学损伤的主要组织化学改变。方法：对动物模型进行视网膜超微结构观察、丙二醛含量测定、细胞色素氧化酶及镁激活三磷酸腺苷酶活性的组织化学观察。结果：超微结构发现光照后6小时，光感受器细胞核肿胀，内节线粒体肿胀，外节水肿，视网膜色素上皮(retinal pigment epithelitis,RPE)顶端微绒毛消失，溶酶体增多，6天反应加重。14天光感受器细胞及内节已基本正常，外节再生但盘膜排列稀疏，RPE顶端出现微绒毛。观察到光照后6小时及６天，视网膜细胞色素氧化酶及镁激活三磷酸腺苷酶活性下降，丙二醛含量增高，至１４天均有所恢复。结论：脂质过氧化使光感受器细胞膜系统损伤崩解，引起细胞超微结构及酶活性改变，可能与视网膜光化学损伤的发病有关。 (中华眼底病杂志,1998,14:38-40)     

视网膜光损伤机制中NOS作用的实验研究

目的　探讨一氧化氮合酶(nitricoxidesynthase ,NOS)活性异常变化在视网膜光损伤发生机制中的作用。方法　60只SD大鼠随机分为5组,1组为正常对照,其它4组以持续强光照射6小时造成视网膜光损伤,并按光照后1天和6天两个观察点各分为实验组与空白对照组,实验组大鼠给予左旋硝基精氨酸治疗,并分别测定比较各组大鼠视网膜外核层厚度以及NOS和超氧化物歧化酶(superoxidedismutase ,SOD)的活性。结果　光损伤后第1天和第6天空白对照组大鼠视网膜内NOS的活性均异常升高,SOD活性均显著下降,视网膜外核层厚度进行性减小;实验组大鼠视网膜内NOS的活性低于正常水平,SOD活性和视网膜外核层厚度均显著高于相应的空白对照组。结论　过度光照后视网膜内NOS活性持续异常升高是视网膜氧化损伤的重要原因并与感光细胞退行性变性的发生密切相关。     

Control of peroxyntrite -induced production of inducible nitric oxide synthase isoforms and antagonism of cholecystokinin octapeptide-8 in retinal pigment epithelial cells in vivo

· AIM: To explore if peroxyntrite (ONOO-) induced iNOS via Fas/ Fas/L pathway in diabetic rats and the effection of cholecystokinin octapeptide-8 (CCK-8) as therapeutic agent for decrease diabetic retinopathy. · METHODS: Thirty-six rats were taken as control group, seventy two were given (streptozotocin) STZ (45mg/kg) and then divided into ONOO-group and CCK-8 group (peritoneal injection CCK-8). STZ-induced diabetic rats were treated with CCK-8 for 60 days. Western blotting analysis, DNA ladder, RT-PCR, immunohistochemistry and flow cytometry were used for determining the expression of nitrotyrosine (NT, the foot print of ONOO-); apoptosis and inducible nitric oxide synthase (iNOS) mRNA as well as Fas/Fasl signal transduction in RPE cells. · RESULTS: Both RPE cells in ONOO- and CCK-8 group developed apoptosis and expressed NT, iNOS mRNA and Fas/Fasl. But latter delayed the all changes in a time-dependent manner compared with control and ONOO- group (P<0.001). iNOS and Fas/Fasl were up-regulated and associated with an increase of expression of ONOO-in vivo. · CONCLUSION: The study suggested that apoptosis of RPE was partly induced by ONOO- may be the new way of oxidative damage to the RPE cells. CCK-8 decreased RPE cells apoptosis partly induced by ONOO- and is a potential drug for therapy of diabetic retinopathy. The mechanism of CCK-8 dealing with RPE cells may be related to its direct inhibition of the formation of iNOS to produce ONOO- and antagnism of damage of ONOO- to RPE cells. ·     

Ascorbate treatment prevents accumulation of phagosomes in RPE in light damage.

In dark-reared albino rats, exposure to 2 or 3 hr of intense light interrupted by 2 hr dark periods resulted in extensive degeneration of photoreceptor cells and degeneration of the retinal pigment epithelium (RPE). Ascorbate (ie, vitamin C) administration prior to light exposure protected photoreceptors and the RPE from light damage. In the present study, ascorbate-treated and untreated rats were exposed to various cycles of intermittent light. Immediately after this light exposure, phagosome frequency in the RPE was morphologically evaluated in comparable 50 microns sections. In untreated rats, exposure to 2 or 3 hr of intermittent light resulted in a five- to sixfold increase in phagosome density compared to unexposed controls. In contrast, no increase in phagosome density was observed in ascorbate-treated rats. In these animals, under all lighting regimens, phagosome levels remained essentially identical to those in rats not exposed to light. After a single nondamaging light exposure, phagosome density remained at the level of dark controls in ascorbate-treated and untreated rats. These results indicate that phagosome frequency may serve as an index for light damage and that the protective effect of ascorbate may be linked to its capacity to prevent rod outer segment shedding and phagocytosis under intense light conditions.     

Neurotrophic Factors, Cytokines and Stress Increase Expression of Basic Fibroblast Growth Factor in Retinal Pigmented Epithelial Cells

Basic fibroblast growth factor (bFGF) and FGF receptors have been localized to photoreceptors and retinal pigmented epithelium (RPE), but the function of bFGF in adult retina and RPE is unknown. Exogenous bFGF has a neuroprotective effect in retina and brain and its expression is increased in some neurons in response to cytokines or stress. In this study, we investigated the effect of light, other types of stress, neurotrophic factors, and cytokines on bFGF levels in cultured human RPE.Some agents that protect photoreceptors from the damaging effects of constant light, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor, and interleukin-1β, increase bFGF mRNA levels in RPE cells. Intense light and exposure to oxidizing agents also increase bFGF mRNA levels in RPE cells and cycloheximide blocks the increase. An increase in bFGF protein levels was demonstrated by ELISA in RPE cell supernatants after incubation with BDNF or exposure to intense light or oxidizing agents. These data indicate that bFGF is modulated in RPE cells by stress and by agents that provide protection from stress and support the hypothesis that bFGF functions as a survival factor in the outer retina.     

Evidence that a-wave latency of the electroretinogram is determined solely by photoreceptors

PURPOSE: To identify the retinal cells that determine the a-wave latency of rats. METHODS: Electroretinograms (ERGs) were recorded from the rod-dominated (0.85% cones) retinas of Long-Evans rats following an intravitreal injection of 1 microL of 40 mM 2-amino-4-phosphonobutyric acid to block the activity of the ON pathway of the second order retinal neurons. ERGs were also recorded following an intraperitoneal injection of sodium iodate to destroy the retinal pigment epithelial (RPE) cells. Damage to a large area of the retina was produced by constant light exposure, and focal damage to the retina was induced by argon laser photocoagulation. The effects of age and anesthesia level on the a-wave latency were also determined. RESULTS: Blocking the activity of the ON pathway of the second order retinal neurons did not alter the a-wave latency, and destroying the RPE cells also did not alter the a-wave latency. Damage to a large area of the retina resulted in prolonging the latency but focal retinal damage did not alter the a-wave latency. The a-wave latency was longer in young rat pups but was adult-like by 18 days. The level of anesthesia had no effect on the latency except at very deep stages. CONCLUSIONS: The a-wave latency is determined solely by the activity of the photoreceptors. A prolonged latency would indicate that the photoreceptors are damaged over a large area of the retina.     

Glutathione peroxidase induced in rat retinas to counteract photic injury

PURPOSE: To examine the hypothesis that glutathione peroxidase (GPX) is induced at different time points after retinal exposure to light and localizes in different retinal cells. METHODS: The rats were kept in cyclic light for 2 weeks before the experiments. The animals were maintained in 12-hour light-dark cycles, before and after exposure to intense white fluorescent light, for as long as 24 hours and then returned to cyclic light. Expression of GPX was measured by immunohistocytochemistry and Western and Northern blot analyses. Light-induced retinal damage was determined by the thickness of the outer nuclear layer (ONL) thickness in relation to total retinal thickness. RESULTS: GPX labeling did not appear in the photoreceptor inner segments, and slight labeling was observed in the photoreceptor outer segments or the retinal pigment epithelial (RPE) cells in the normal retina kept in cyclic light. In retinal specimens maintained in light for 12 and 24 hours, GPX labeling was induced in the photoreceptor outer segments and RPE cells. High expression of GPX in the RPE was sustained until day 7 after challenge. In contrast, GPX expression in the photoreceptor outer segments decreased on day 1 and disappeared on days 3 and 7 after exposure. Intense GPX labeling was seen from the internal limiting membrane to the ganglion cell layer. GPX labeling was constantly localized in both high-intensity white light and cyclic conditions, suggesting no induction of GPX in those areas. In addition, GPX labeling was apparent at the posterior retinal pole but not at the peripheral retina. We observed marked upregulation of GPX mRNA in rats kept in high-intensity white light. One, 3, and 7 days after exposure to high-intensity white light, there was a significant difference (P < 0.0001) between the control and experimental groups in the ratio of the outer nuclear layer thickness to the entire retina. CONCLUSIONS: GPX was induced at different time points after exposure to high-intensity white light and localized in different retinal cells. Changes in expression of GPX after exposure to light may be related to the difference in susceptibility of the retina to damage by light.     

In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonic stem cells

PURPOSE: To determine whether primate embryonic stem (ES) cell-derived pigment epithelial cells (ESPEs) have the properties and functions of retinal pigment epithelial (RPE) cells in vitro and in vivo. METHODS: Cynomolgus monkey ES cells were induced to differentiate into pigment epithelial cells by coculturing them with PA6 stromal cells in a differentiating medium. The expanded, single-layer ESPEs were examined by light and electron microscopy. The expression of standard RPE markers by the ESPEs was determined by RT-PCR, Western blot, and immunocytochemical analyses. The ESPEs were transplanted into the subretinal space of 4-week-old Royal College of Surgeons (RCS) rats, and the eyes were analyzed immunohistochemically at 8 weeks after grafting. The effect of the ESPE graft on the visual function of RCS rats was estimated by optokinetic reflex. RESULTS: The expanded ESPEs were hexagonal and contained significant amounts of pigment. The ESPEs expressed typical RPE markers: ZO-1, RPE65, CRALBP, and Mertk. They had extensive microvilli and were able to phagocytose latex beads. When transplanted into the subretinal space of RCS rats, the grafted ESPEs enhanced the survival of the host photoreceptors. The effects of the transplanted ESPEs were confirmed by histologic analyses and behavioral tests. CONCLUSIONS: The ESPEs had morphologic and physiological properties of normal RPE cells, and these findings suggest that these cells may provide an unlimited source of primate cells to be used for the study of pathogenesis, drug development, and cell-replacement therapy in eyes with retinal degenerative diseases due to primary RPE dysfunction.     

可见光对培养的人视网膜色素上皮细胞内碱性成纤维细胞生长因子、成纤维细胞生长因子受体1、B细胞淋巴瘤/白血病-2基因及 caspase-3的影响

目的　观察可见光诱导的培养人视网膜色素上皮 (RPE)细胞凋亡中 ,RPE细胞内碱性成纤维细胞生长因子 (bFGF)、成纤维细胞生长因子受体 1(FGFR1)、B细胞淋巴瘤 /白血病 2基因(bcl 2 )及caspase 3的表达变化。方法　建立可见光诱导培养的人RPE细胞凋亡的模型 ,通过细胞免疫组织化学、逆转录聚合酶链反应 (RT PCR)、酶联免疫测定等方法 ,对培养的人RPE细胞内bFGF和其受体FGFR1的mRNA表达变化、Bcl 2表达改变以及caspase 3的活性变化进行了检测。 结果(1)光照后 6及 12h ,培养的人RPE细胞胞质内Bcl 2蛋白的表达有一定的下降趋势 ,但与无光照组之间差异无显著性意义 (P >0 0 5 )。光照后 2 4h ,Bcl 2蛋白表达下降明显 (P <0 0 1)。bcl 2mRNA表达与上述蛋白表达变化相似 ,但早在光照 12h后已能检测到其表达下降。 (2 )光照后 6hbFGF及FGFR1的表达尚无明显改变 (P >0 0 5 ) ,随着时间延长 ,其表达增加的差异具有显著意义(P <0 0 5 )。(3)光照后caspase 3活性随时间延长而明显增加 ,与未光照时比较 ,光照结束后 6、12及2 4h活性差异有显著意义 (P <0 0 5 )。 12和 2 4h间差异无显著意义 (P >0 0 5 )。结论　可见光照诱导培养的人RPE细胞凋亡时 ,RPE细胞内bcl 2表达下降 ,内源性bFGF及FGFR1上调 ,caspase 3