正常人视网膜色素上皮细胞扫描电镜观察

视网膜色素上皮细胞具有屏障、输送营养等重要作用,近年来,人们采用光镜和透射电镜等观察手段,对中国人正常视网膜色素上皮细胞超微结构有了较祥细的     

氧化损伤对体外培养人视网膜色素上皮细胞PEDF的影响

目的研究氧化损伤对人视网膜色素上皮(retinal pigment epithelial,RPE)细胞表达色素上皮细胞衍生因子(pigment epithelium derived factor,PEDF)的影响。方法体外培养人RPE细胞,加入浓度为600μmol.L-1H2O2分别作用不同时间(2h、8h、24h),采用免疫细胞化学法检测PEDF蛋白的表达,逆转录聚合酶链式反应(RT-PCR)法检测PEDF mRNA。结果免疫细胞化学染色对照组即有PEDF的阳性表达,而氧化损伤2h、8h、24h PEDF蛋白阳性表达量逐渐减少,染色由棕黄色变为黄色。各时间段两组结果比较,差异均具有统计学意义(P<0.05)。RT-PCR检测PEDF mRNA量与PEDF蛋白表达变化一致。结论氧化损伤能促使人RPE细胞PEDF表达下调,并在一定范围内与作用时间有关。     

颜色光对体外培养人视网膜色素上皮细胞的光损伤

目的　在体外培养人视网膜色素上皮细胞的基础上 ,比较蓝、绿、黄光对视网膜色素上皮的损伤。　方法　取第二代融合细胞作为光照射对象 ,用强度为 5 0mW /cm2 的蓝、绿、黄光进行不同时间的光照射 ,通过光镜、电镜检查以及细胞增殖抑制实验比较损伤的差异。　结果　三种颜色光均可引起体外培养的视网膜色素上皮细胞光损伤 ,蓝光的损伤作用最强 ,绿光和黄光的损伤作用近似。　结论　在蓝、绿、黄光所致的视网膜色素上皮光损伤中 ,蓝光的损伤作用最强。     

YVAD-cmK及DEVD-CHO对培养的人视网膜色素上皮细胞光损伤的影响

目的 观察半胱天冬酶(caspase-3)选择性抑制剂天冬氨酸-谷氨酸-缬氨酸-天冬氨酰胺醛(Asp-Glu-Val-Asp-CHO,DEVD-CHO),及caspase-1选择性抑制剂酪氨酸-缬氨酸-丙氨酸-天冬氨酰胺(Tyr-Val-Ala-Asp-cmK,YVAD-cmK)对可见光诱导的培养的人视网膜色素上皮(RPE)细胞凋亡的影响。方法 用(2000±500)lx的白色荧光灯光源,照射培养的人RPE细胞。在培养液中加入YVAD-cmK和DEVD-CHO,利用荧光素标记的连接素V/碘化丙锭双染色流式细胞测定观察RPE细胞的凋亡情况。结果 本研究模型下,DEVD-CHO预处理组的细胞凋亡及坏死与无光照组相比,差异无显著性(P>0.05),与光照组及YVAD-cmK预处理组相比,细胞存活数增高,差异有显著性(P<0.05)。而YVAD-cmK组与无光照组比较,其结果与单纯光照组一样,表现出细胞的明显损伤(P<0.05)。结论 可见光诱导培养的人RPE细胞凋亡的发生机制中,可能不涉及casrpase-1的作用。外源性的caspase-3抑制剂(DEVD-CHO)对RPE细胞光性损伤有一定的保护作用。     

视网膜色素上皮细胞相关细胞因子

很多眼底疾病与视网膜色素上皮 (retinalpigmentepithelium ,RPE)细胞功能改变有关。近期大量研究表明RPE细胞功能异常及分泌多种细胞因子参与这些疾病。检测这些细胞因子可反映RPE细胞的功能状态。对RPE参与的多种眼底疾病的研究和治疗提供了新的思路和手段     

牛磺酸对氧化损伤人视网膜色素上皮细胞 caspase-3 表达的影响

目的探讨牛磺酸在氧化损伤的人视网膜色素上皮(retinal pigment epithelium,RPE)细胞凋亡中的作用机制。方法体外培养的RPE细胞加入600μmol.L-1H2O2,制备氧化损伤模型,加入牛磺酸共同孵育,采用免疫组织化学法检测caspase-3的表达,ELISA检测上清液中caspase-3的浓度。结果Caspase-3在正常的RPE细胞中未见明显表达,在氧化损伤组其表达位于胞核,为特异性黄色着色,且随时间增加而表达增强。牛磺酸保护组caspase-3表达规律与氧化损伤组相似,但较相应氧化损伤组明显减少,差异具有显著统计学意义(P<0.05)。结论Caspase-3参与视网膜氧化损伤凋亡的发生,牛磺酸对视网膜氧化损伤可能有保护作用。     

黄芪多糖对过氧化氢诱导人视网膜色素上皮细胞氧化损伤的保护作用

目的观察黄芪多糖(astragalus polysaccharide,APS)对过氧化氢诱导的人视网膜色素上皮细胞(retinal pigment epithelium,RPE)氧化损伤的保护作用。方法培养的人RPE细胞系ARPE-19分为正常对照组、过氧化氢损伤组和不同浓度(1000 mg·L-1、500mg·L-1)APS干预组,正常对照组不作任何处理,过氧化氢损伤组加入200μmol·L-1过氧化氢,APS干预组加入不同浓度(1000 mg·L-1、500 mg·L-1)APS后加入200μmol·L-1过氧化氢。用倒置荧光显微镜观察各组细胞形态变化;MTT检测各组细胞活性;DAPI染色观察各组细胞核形态学变化;实时细胞电子分析系统(real-time cell electronic sensing system,RT-CES)实时记录细胞生长状态;应用caspase-3活性检测试剂盒测定各组细胞中caspase-3的活性。结果过氧化氢损伤组细胞皱缩,悬浮细胞增多,APS干预后细胞状态改善。MTT检测结果显示:1000 mg·L-1、500 mg·L-1APS孵育24 h后细胞存活率分别为(99.86±3.64)%和(101.03±3.52)%,与正常对照组(100%)相比,差异均无统计学意义(均为P>0.05),过氧化氢损伤组细胞存活率为(56.49±2.30)%,与正常对照组相比显著降低(P<0.01),1000 mg·L-1、500 mg·L-1APS干预后,细胞存活率升高到(88.14±2.97)%和(80.75±3.13)%,与过氧化氢损伤组相比差异均有显著统计学意义(均为P<0.01)。DAPI染色显示正常细胞核均匀淡染,过氧化氢损伤组出现强荧光点和致密的细胞核,APS处理后凋亡程度减轻。RT-CES显示,APS处理可以减少过氧化氢造成的细胞损伤。Caspase-3检测发现过氧化氢造成细胞caspase-3水平升高,APS处理后caspase-3水平下降。结论 APS可以抑制过氧化氢诱导的人RPE细胞系ARPE-19的凋亡,这为APS的临床应用提供了一定的实验基础。     

培养人视网膜色素上皮细胞机械损伤后IL-8的表达

目的探讨视网膜色素上皮(retinal pigment epithelium,RPE)细胞受机械损伤后IL-8表达情况。方法取在6孔培养板内培养的RPE细胞,并建立机械损伤模型。待细胞铺满融合后,每孔刮除相同面积的细胞,72h后收集培养上清液,采用ELISA试验检测培养上清液中IL-8的表达,同时用免疫组织化学方法检测RPE细胞内IL-8的表达。结果 RPE细胞在基础状态下,免疫组织化学法检测细胞内IL-8的表达,几乎不着色。经过机械损伤刺激充分反应后,在损伤边缘的RPE细胞内IL-8蛋白质的表达呈阳性,胞浆出现浓淡不一的棕黄色着色。ELISA检测结果显示,体外培养RPE细胞在基础状态下可表达少量的IL-8,在经过机械损伤反应后,IL-8表达量显著增加,3次检测中对照和刮伤模型上清液中IL-8的浓度分别是105×10-12kg.L-1、965×10-12kg.L-1,108×10-12kg.L-1、990×10-12kg.L-1,100×10-12kg.L-1、960×10-12kg.L-1,两者间有显著差异(P<0.01)。结论体外培养RPE细胞经过机械损伤反应后,IL-8的分泌和表达显著增加。提示在视网膜脱离前及脱离过程中,RPE细胞受到通过视网膜神经上皮层传导过来的机械牵拉力的作用后在损伤的修复过程中,可能会诱导IL-8的产生,从而有助于启动RPE细胞的移行及早期的炎症反应,导致PVR的发生。     

Scanning and transmission electron microscopic findings during RPE wound healing in vivo

Objective: To examine the scanning (SEM)and transmission (TEM) electron microscopic features of an in vivo rabbit model of retinal pigment epithelial (RPE) wound healing. Methods: Hydraulic debridement of the RPE was performed in one eye of each of 35 pigmented rabbits using a pars plana vitrectomy approach. Five of the 35 eyes were examined by either SEM or TEM on each of the following postoperative days: 0, 2, 4, 7, 14, 28 and 56. Results: TEM revealed that hydraulic RPE debridement results in only focal damage to the RPE basement membrane portion of Bruch‘s membrane and that this damage is repaired by day 7 without ultrastructural sequelae. SEM and TEM disclosed that the RPE cells at the margin of the debrided bed become flattened and enlarged and evolve a cytoskeletal reorganization with altered apical-basal polarity consistent with the development of a migrating phenotype. This is followed by gradual restoration to a more normal stationary RPE phenotype after initial closure (reepithelialization) of the RPE defect on day7. RPE hyperplasia also occurs and may contribute to this repair process. Tight junctions are re-established among the apical surfaces of monolayered and multilayered RPE cells by day 7, coinciding with the restoration of the blood outer retinal barrier. Conclusion: Hydraulic debridement of the RPE in vivo is a useful investigational model that provides important insight into the pathogenesis of outer retinal disorders and their treatment with suchtechniques as submacular surgery or RPE transplantation. This revised version was published online in August 2006 with corrections to the Cover Date.     

New ultrastructure of rat RPE cells: basal intracytoplasmic tubules

Retinal pigment epithelial cells have prominent basal folds facing Bruch's membrane. In addition to folds I have observed intracytoplasmic tubules 60-90 nm in diameter in the basal cytoplasm of rat pigment epithelial cells. The tubules arise from the basal plasma membrane and open to the extracellular space. The tubules are most evident when intravenous horseradish peroxidase is used as a tracer. The tracer leaks out of the fenestrated choriocapillaris, into the extracellular space of the pigment epithelium and into the tubules. Electron microscopy at 1000 KV (High Voltage Electron Microscopy) confirms the tubular nature of these structures and their continuity with folds or the plasma membrane facing Bruch's membrane. The tubules are also observed in tissue not infiltrated with peroxidase. Morphometry shows that the tubules occupy about 21% of the surface area of the basal plasma membrane. Tubules appear plentiful where folds are reduced, and reduced where folds are plentiful; the tubules may be a different conformation of the normally slit-like fold extracellular space. The tubules are observed in all quadrants of the retina; centrally and peripherally; in young and adult rats and in pigmented and albino rats. The tubule's function may be linked to that of the folds, from which many of them arise.     

神经生长因子对人胚胎视网膜色素上皮细胞生长及其DNA合成的影响

目的　探讨神经生长因子 (NGF)对体外培养的人胚胎视网膜色素上皮 (RPE)细胞的促增殖作用及DNA合成的影响。方法　分别用MTT法测定加入NGF 10 0、2 0 0、30 0 μg/L 48h后人RPE细胞数量的改变和核酸蛋白分析仪测定加入NGF 5 0、10 0、2 0 0、30 0、40 0 μg/L 48h后人RPE细胞DNA质量浓度的变化。 结果　 10 0、2 0 0、30 0 μg/LNGF作用 48h后其细胞增殖的A值分别为 ( 0 2 137± 0 0 2 33)、( 0 2 188± 0 0 181)、( 0 2 32 2± 0 0 16 4) ,与对照组 ( 0 1897± 0 0 15 2 )相比 ,差异有显著性 (q test ,P <0 0 5 ) ;10 0、2 0 0、30 0、40 0 μg/LNGF作用 48h后其细胞DNA质量浓度分别为 ( 981 2 2 0 4± 12 3 5 35 7)、( 1375 8484± 2 44 4 718)、( 16 5 8 70 71± 176 9381)、( 2 35 3 0 86 3± 6 0 9 90 6 4) μg/ml ,与对照组 ( 6 6 6 8188± 14 1 330 2 ) μg/ml相比差异有显著性 (q test,P <0 0 5 )。结论　NGF可促进人RPE细胞增殖及促进细胞DNA合成。     

西洋参茎叶总皂甙对培养人胚视网膜色素上皮细胞增生的影响

目的 研究西洋参茎叶总皂甙(PQS)对人视网膜色素上皮细胞(RPE)增生的抑制作用。方法 通过活细胞计数法与MTT比色法分别检测不同质量浓度(10μg／ml～500μg／ml)PQS和PQS(200μg／ml)在不同作用时间(6～120h)对RPE增生及代谢的影响。结果 PQS组细胞增殖受抑制，细胞生存率下降，400μg／ml抑制作用最强；其抑制效应在6h出现，72h达高峰。结论 PQS可能通过阻滞钙通道、干扰RPE代谢对RPE增殖具有剂量和时间依赖方式的抑制作用。     

Interpretation of some specular microscopical images of insulted corneal endothelium by scanning electron microscopy

The interpretation of clinical specular microscopical images of abnormal corneal endothelium remains a problem in many instances when the relief mode is negative. This is primarily because different cellular changes occurring simultaneously present similar appearances. In this paper is compiled a number of specular images of small, dark (non-reflecting) cell changes in the endothelium which resemble one another and clinical examples, that have been induced in rabbit eyes by various means. Specific cells showing these changes have been relocated by scanning electron microscopy and orientated to match specular photomicrographs taken before fixation. This has allowed precise comparison of their specular and scanning electron microscopical appearances and the interpretation of the former by the latter. It is found that reversible changes are expressed as an excess of microvilli on the cells and minor inclination of the posterior cell membrane from the perpendicular with the axis of the microscope, and irreversible changes are due to cell fragmentation or total loss with exposure of Descemet's membrane to the aqueous humour. Re-examination of specular photomicrographs with this knowledge in mind indicates that very subtle differences in the specular images often betray the true nature of the cell change. An exception to the rule is included.     

红景天对培养人视网膜色素上皮细胞增生和DNA合成的抑制作用

目的 研究红景天对体外培养视网膜色素上皮细胞增生的抑制作用及可能机制。方法 通过活细胞计数法与MTT比色法分别检测不同质量浓度(500、400、300、200、100、50、10μg／mL)红景天和红景天200μg／mL在不同作用时间(6～120h)对RPE细胞增生及代谢的影响。结果 红景天组细胞增生受抑制并出现细胞脱落，红景天400μg／mL具有最强的抑制效应，其明显抑制效应在6h即出现，72h达高峰。红景天组L4值日明显低于对照组，RPE细胞生存率下降。结论 红景天可能通过干扰RPE细胞代谢对RPE细胞增殖具有剂量依赖和时间依赖方式的抑制作用。     

人视网膜色素上皮细胞NF-kB的表达

目的探讨核因子闎(NF-кB)在体外培养的人视网膜色素上皮(human retinal pigment epithelium,hRPE)细胞中的基础表达以及吡咯二硫代氨基甲酸乙酯(pyrollidi ne dithiocarbamate,PDTC)、IL-1β对NF-кB表达的影响. 方法体外培养的hRPE细胞经同步化后分2组分别加药(1)无PDTC组分别加入IL-1β、生理盐水(用于检测NF-кB在hRRE中的基础表达);(2)PDTC组分别加入IL-1β、生理盐水(用于检测NF-кB在PDTC预处理后hRPE中的表达).免疫荧光抗体染色、流式细胞计数法(flow cytometry, FCM)测定上述2组标本中hRPE的NF-кB的表达率. 结果 NF-кB在hRPE中的基础表达率为8.05 %,IL-1β作用后表达率升高到30.26%; hRPE细胞经PDTC预处理后,NF-кB的表达率下降为3.74%,加入IL-1(10 υg·L-1)作用后表达率为3.66%. 结论 IL-1β可以显著提高NF-кB在hRPE细胞中的表达;PDTC可以显著降低体外培养的hRPE中的NF-кB表达率,PDTC亦可显著抑制由IL-1β诱导的NF-кB的活化过程.     

Scanning electron microscopy of the frog lens

We have studied the morphology of the frog lens by scanning electron microscopy (SEM). By using fixative solutions with various buffer concentrations, we have been able to find a proper combination which significantly reduces tissue shrinkage during fixation. This has allowed us to control the shrinkage and cracking of lens which were previously thought to result from the process of critical point drying. We can now examine by SEM the surface morphology of practically every lens cell in a single antero-posterior plane. Thus, for the first time the membrane surfaces of the lens epithelial cells (central, pregerminative, germinative and transitional zones), the elongating fibers, fibers through the varying depths of the cortex, and the aged nuclear fibers can all be examined and compared in a single lens. The ability to prepare the entire lens for morphological examination with minimized preparative artifacts is important to further studies where stereological measurements can be made to quantitate for example, total membrane surface area and the size of the extracellular space at various locations within the lens. A quantitative estimation of these parameters will result in a more accurate analysis of the electrical properties of the lens.     

丝状角膜炎的共聚焦显微镜观察

目的：应用活体激光共聚焦显微镜（ IVCM）观察丝状角膜炎患者丝状物的组成结构及位置特点,分析角膜丝状物的形成机制。方法收集深圳市眼科医院2014年6月至2016年1月收治的丝状角膜炎患者48例（60只眼）,详细记录患者的病史和相关检查。采用共焦显微镜观察角膜丝状物的组成结构和角膜丝状物附着处的角膜结构。结果活体激光共聚焦显微镜检查显示,丝状物起自前弹力层,由上皮细胞、炎性细胞、黏液、螺旋状条形核心构成,核心结构中亦含有上皮细胞、炎性细胞及纤维状组织。结论丝状角膜炎丝状物的共聚焦表现特点可以为临床治疗方法的选择提供影像的参考依据。