鸡胚视网膜细胞RNA合成的放射自显影研究

目的　研究视网膜细胞发生过程中的形态变化与RNA合成代谢的关系。方法应用光镜放射自显影技术 ,对 1、2、3、4、7d鸡胚视网膜氚标尿嘧啶的分布进行了观察。结果　视杯各部的氚标尿嘧啶银颗粒数均随胚龄增加而增多 ,以前部最为显著 ;标记细胞的银颗粒主要分布在细胞核内。结论　伴随视网膜细胞的分化成熟 ,RNA的合成数量和区域分布表现为明显的动态变化。     

大鼠尾芽胚视杯干细胞的分布与特征

目的探索视杯干细胞在大鼠尾芽胚(第12.5天胚龄)的分布与特征。方法采用免疫组织化学技术，检测视杯干细胞在大鼠尾芽胚(第12.5天胚龄)视杯组织中的分布；分离视杯细胞，体外无血清培养，应用免疫细胞化学技术分析其增生能力以及血清诱导分化前后CHX10和多种成熟视网膜细胞特异性标记蛋白的表达，以了解这一发育时期视杯组织的分化特点。结果大鼠尾芽胚(第12.5天胚龄)的视杯干细胞主要分布在视杯的内外层和边缘层，不表达成熟视网膜细胞特异性标记蛋白。从尾芽胚视杯中分离出的细胞具有单细胞克隆能力，CHX10表达阳性，血清诱导后表达多种成熟视网膜细胞特异性标记蛋白：Thy1.1、神经胶质纤维酸性蛋白(GFAP)、蛋白激酶C(PKC)α和rhodopsin。结论大鼠尾芽胚(第12.5天胚龄)视杯主要由未分化的细胞组成，视杯干细胞的分布集中在视杯内层和边缘层。体外培养的视杯干细胞增生能力强，经诱导分化后表达多种成熟视网膜细胞特异性标记蛋白。(中华眼底病杂志,2005,21:159-162)     

人视网膜星形胶质细胞发育及起源的研究

背景 视网膜星形胶质细胞是视网膜主要的神经胶质细胞,其起源及演变过程一直是国内外研究的热点和难点. 目的 探讨人胚胎眼视网膜星形胶质细胞的起源及发育.方法 收集33例自愿终止妊娠的流产人胚胎眼标本,其中8 ～12孕周者20例,15～ 17孕周者2例,19～ 23孕周者4例,25～ 28孕周者4例,30 ～32孕周者3例.对眼球壁切片进行常规组织病理学检查以观察不同胚龄人视网膜发育的形态学变化,分别采用免疫组织化学法及免疫荧光法动态观察不同胚龄人视网膜星形胶质细胞起源位点及发育过程中胶质纤维酸性蛋白( GFAP)表达的变化.结果 人胚6～7周视杯处于视网膜分层发育阶段,9周时视杯内层原无细胞层出现分化不成熟的圆短梭形细胞；胚龄15周时视网膜主要层次可见,分化的细胞增加,但未发现GFAP阳性细胞；胚龄19周视网膜可见梭形细胞从返折部原始神经上皮迁出,并可见这些细胞中GFAP呈阳性表达；胚龄25～ 26周后极部视网膜可见GFAP表达阳性的梭形细胞,这些细胞围绕视网膜血管分布,与血管壁联系密切,邻近锯齿缘处的视网膜内层可见表达GFAP的星形或梭形细胞与睫状体非色素上皮相连,但锯齿缘稍后与赤道区之间并未见GFAP阳性细胞；胚龄28周,视网膜星形胶质细胞呈典型的星状,其突起伸达视网膜内网状层. 结论 人视网膜星形胶质细胞至少存在3个起源位点,即血管前体细胞/周皮细胞、视盘旁原始神经上皮及邻近锯齿缘的睫状体无色素上皮.     

视网膜色素上皮与有关常见病

视网膜是眼球最重要的眼内组织,是视觉系统中的前哨。组织学上共分为十层,最外层为视网膜色素上皮,起源于视杯外层;其余九层为神经视网膜,由视杯内层分化而来。视网膜色素上皮(RPE)与神经视网膜(简称视网膜)相比,组织结构上看来相对简单,但功能上却十分复杂,其对视觉的影响至关紧要,必须予以重视。近代视觉研究中,视网膜色素上皮是众所关注的课题之     

合理选择视网膜电图检查指标 客观了解内层视网膜的功能

视网膜的解剖结构和生理功能非常复杂,目前临床上视网膜功能的客观评估主要依靠视网膜电图(ERG).对主要累及外层视网膜的疾病,ERG可以准确判断病变所处的组织学部位,但对主要位于内层视网膜,即视细胞突触以后的视网膜病变,视觉电生理检查的辨识度尚远不及于外层视网膜,这是因为内层视网膜神经传导通路较长、内层视网膜也受外层视网膜视觉信号传入变化的影响以及视杆系统和视锥系统的反应存在相互影响,因此客观了解内层视网膜的功能是一个难点.近20年来,一些新的能够用于检测内层视网膜功能的ERG检测技术,如振荡电位(OPs)、ON-OFF反应、明视负波反应(PhNR)、暗视阈值反应(STR)等已用于临床,这些ERG指标主要起源于双极细胞、无长突细胞和视网膜神经节细胞,是评估内层视网膜疾病的有用工具.临床眼科医师应充分了解这些ERG检测指标在内层视网膜疾病诊疗中的临床意义,从而更好地指导临床诊疗工作.     

对视网膜细胞蛋白合成的加龄变化研究

应用光镜放射自显技术的胎生期至生后一年鼠的视网膜各层细胞的蛋白合成变化进行了研究，结果表明，视杆，视锥细胞生后至一年始终保持着旺盛的蛋白合成功能，胎生期至生后一周，视杆，视锥细胞处于不成熟的分化阶段，视网膜各层细胞的胞核及胞质内切可见记颗粒，生后第一天其标记率出现第一个高峰，视杆，视锥和双极细胞分化成熟后，可见标记颗粒主要分布在视细胞的胞体，内节及双极细胞的胞质内，且视细胞的标记颗粒多于双极细胞，     

视网膜血管的解剖学研究

视网膜的血液供给,内层和外层不同。即从视网膜色素上皮细胞层到外颗粒层,是通过脉络膜毛细血管的渗透扩散而来。从外丛状层到神经纤维层,由视网膜中央动脉分支供给。视网膜中央动脉,在视神经乳头部分为4支小动脉,并进一步分为2支,最后形成毛细血管。视网膜静脉系统也和动脉几乎平行走行,4支小静脉汇集在视乳头成为视网膜中央静脉。除锯齿缘部以外,动脉和静脉没有吻合。小动静脉存在于神经纤维层,像长短不一的藤从藤棚垂下,其支配下的毛细血管网分布在神经纤维层到神经节细胞层;内丛状层到内颗粒层;内颗粒层到外丛状层的3层。此外,在视乳头周围还有从视乳头伸向     

尿激酶对组织培养人眼小梁细胞的影响

为检验尿激酶在临床应用时对小梁细胞有无不良影响。本研究应用组织培养人眼小梁细胞，观察不同浓度尿激酶对细胞形态的影响，并测定其对细胞DNA合成时氚（标胸腺嘧啶核着（3H-TdR）掺入的量，来判断药物对细胞增殖功能的影响。结果示5000u／ml浓度作用6小时细胞即出现胞突回缩、胞体皱缩等变化，至48小时细胞死亡；5000和500u／ml浓度均显著抑制DNA合成。提示在应用尿激酶冲洗前房积血时，一定要冲洗干净残留药物，以免对小梁细胞造成损害。     

视网膜下人工假体植入后视网膜变化的研究进展

视网膜下人工假体是目前关于视网膜色素变性和老年性黄斑变性等视网膜外层退行变性疾病研究中的一种新的治疗方法。它旨在利用植入的微电极器替代变性的光感受器细胞,经光-电信号转换,对视网膜内层残留的双极细胞和神经节细胞产生电刺激,进而通过正常的视路在大脑视皮质诱发视觉反应,从而达到恢复视力的目的。然而视网膜下假体植入后视网膜的结构、功能及蛋白表达等可发生一些变化,有学者还发现视网膜下假体植入后一段时间内视网膜的结构和功能得到改善,推测可能有保护作用的参与,但其来源尚不完全明了。本文主要就视网膜下人工假体植入后视网膜的变化作一综述。     

维拉帕米对培养的人视网膜色素上皮细胞增殖和DNA合成的影响

目的:观察钙通道阻滞剂维拉帕米(verapamil)对培养的人视网膜色素上皮细胞(human retinalpig-ment epithelium,hRPE)增殖和DNA合成的影响。方法:含不同浓度维拉帕米的条件培养液作用于培养的hRPE细胞后,利用细胞计数和MTT比色试验观察维拉帕米对培养的hRPE细胞的作用,同时采用氚标胸腺嘧啶核苷(3H-thymidine,3H-TdR)掺入试验测定维拉帕米对培养的hRPE细胞DNA合成的影响。结果:维拉帕米在实验所设的浓度范围均能抑制培养的hRPE细胞的增殖和DNA合成,在浓度为10,50,100,200μm ol/L时其细胞增殖抑制率分别为:26.72%,37.25%,58.21%,79.54%(P<0.01);cpm值分别为对照组的0.81,0.61,0.52,0.44倍(P<0.01)。结论:维拉帕米对培养的hRPE细胞增殖和DNA合成具有抑制作用。     

Quinolinate. A selective neurotoxin in embryonic and posthatching chicken retinas

Quinolinate (QUIN), an endogenous dicarboxylic amino acid, structurally related to the putative retinal neurotransmitter aspartate, acts as a specific neurotoxin in the chick neural retina. Qualitative analysis of QUIN's neurotoxic effects reveals that sensitivity to the amino acid is first detected in the 9-day-old embryonic chick retina. Nuclei and cytoplasm of some cells in the inner region of the inner nuclear layer and in the ganglion cell layer appear hypochromatic or electron lucent when examined by light or electron microscopy, respectively. Between day 10 and 12, the sensitivity of the embryonic retina to QUIN increases and remains around the day 12 level throughout the remaining embryonic and initial posthatching period. Cells in the inner half of the inner nuclear layer continue to be the most severely affected throughout retinal development, ganglion cells less so. Photoreceptor and most cells in the outer region of the inner nuclear layer remain undamaged. QUIN effects are partially reversible: retinas exposed to QUIN briefly in vitro and then transferred to fresh QUIN-free medium are not as severely affected as those allowed no recovery time. In day 1 posthatching chick retinas, similar patterns of QUIN-toxicity were observed in vitro (0.5-5 mM QUIN; 5-30 min) and in vivo (200-600 micrograms QUIN/eye; 0.5-24 hr following intravitreal injection).     

Studies on the cytodifferentiation of the neuroblasts and visual cells in the chick embryo retina,using the electron-microscopic autoradiography of 3H-thymidine

Summary Studies on the histogenetic analysis of cytodifferentiation of the neuroblast and visual cell in the chick embryo retina were made using the autoradiography of ³H-thymidine. The posterior pole region of the eyeball was observed in all the animals used.The retina in a 4-day-old chick embryo consists exclusively of matrix cells forming the matrix layer. In a 5-day-old chick embryo retina, neuroblasts first differentiated from the matrix cells migrate into the outer part of the matrix layer, forming mantle layer.The matrix cell is a homogeneous epithelial cell containing abundant free ribosomes and a poorly developed cytoplasmic membrane system in the cytoplasm. The characteristic sign of differentiation of the neuroblast is an appearance of elements of rough-surfaced endoplasmic reticulum and an indentation of the nucleus.The primitive visual cell having just lost its ability to synthesize DNA appears just beneath the pigment epithelium in a 7-day-old chick embryo, and all the cells lying beneath the pigment epithelium lose the ability to synthesize DNA at 10 days of incubation. The cytoplasmic process of the matrix cell is in contact with the adjacent one, making an apicolateral junction. When the matrix cell loses its ability to synthesize DNA, a big tentlike process extending over the level of the apicolateral junction appears. This phenomenon is considered to be a sign of differentiation from matrix cell to primitive visual cell, and this big tentlike process containing 2 centrioles is a primordium of the inner segment of the visual cell.     

A microscopic study of herpes simplex virus retinopathy in mice

ICR white mice were inoculated with herpes simplex virus (HSV) type I in the anterior chamber of one eye. Animals were killed at intervals of 2, 4, 6, 8, and 10 days and both eyes were obtained for light and electron microscopic study of retinal changes. HSV retinopathy developed in 42 (91%) of 46 inoculated eyes. Fourteen (88%) of sixteen noninoculated eyes examined after the sixth postinoculation day developed HSV retinopathy. The earliest signs of retinopathy in the inoculated eye were peripheral retinal vasculitis and inflammatory cells throughout the nerve fiber layer on day 2. No virus was found in retinal tissue until day 4, at which time disruption of outer retinal layers (outer nuclear layer and layer of rods and cones) was observed in the peripheral retina. The earliest signs of retinopathy in the noninoculated eye were isolated foci of outer retinal disruption in the posterior retina on day 6. The inflammation accompanying early retinal changes of HSV retinopathy were more severe in the inoculated eye. Electron microscopy of both eyes revealed viral particles in the inner nuclear and ganglion cell layers at the time of outer retinal disruption, but viral particles were seen only rarely in the outer retinal layers at this stage. Early disruption of normal retinal architecture may be due to infection and destruction of Muller cells. The retinopathy progressed in both eyes to total destruction of the retina by day 10. Viral infection of the retinal pigment epithelium occurred, but viral particles were seen only rarely in the underlying choroid. This model may be useful for the study of HSV retinopathy in humans.     

Glutamine fructose-6-phosphate aminotransferase and glycoprotein synthesis in the developing chick eye

Glutamine fructose-6-phosphate aminotransferase is an essential enzyme in the synthesis of glycoproteins and other complex carbohydrates. The specific activity of this enzyme was measured in posterior ocular tissues of the chick embryo from day 6 of development until hatching. The enzyme is present at high concentrations in the early embryo, then decreases 15-fold by day 9 in both retina and retinal pigment epithelium (PE). Thus, aminotransferase activity is highest during the period when retinal cells are migrating and beginning to differentiate; these maturational events may require cell-surface glycoproteins. On day 17-18 of chick embryo development, the neural retina suddenly adheres firmly and irreversibly to the underlying PE. Aminotransferase activity and glucosamine incorporation into retinal macromolecules were examined near this critical time, as were the effects of glycoprotein-synthesis inhibitors upon retinal adhesion. No evidence was found for involvement of complex carbohydrates (on cell surfaces or in the interphotoreceptor matrix, IPM) in formation of retinal adhesion in the chick. Histological studies show an absence of glycosaminoglycans in the IPM of embryonic chicks (at all stages) and of adult chickens, although these compounds are present in mammalian matrix.     

血管内皮生长因子在新生小鼠视网膜的表达

目的 研究血管内皮生长因子（vascular endothelial growth factor,VEGF)与新生小鼠视网膜血管系统形成的内在联系，方法 分别于小鼠出生后3d,1、2、4周以免疫组化观察VEGF在视网膜的表达，并与视网膜铺片和光镜结果相对照。结果 小鼠出生后视网膜血管系统由视盘处呈放射状穿出，逐渐由后极部向周边部，视网膜病理切片显示；出生后3d时，视网膜只有2层，即神经节细胞层和神经母细胞层，内层毛细血管仅限于视网膜后极及中周部，7d时外丛状层形成，出生后14d，视网膜外层毛细血管发育基本完成，出生后28d，视网膜与14d时大致相似，VEGF免疫组化结果显示；出生后3d，阳性信号位于神经节细胞层和神经母细胞层内，外缘，7d时，VEGF明显表达在神经节细胞层，外丛状层和几乎全层内核层，出生后14d及28d。当视网膜血管系统发育基本完成时，VEGF阳性细胞数量和强度都明显减少和减弱。结论 本研究提示，VEGF的表达在空间和时间上与视网膜血管形成密切相关，VEGF的存在对于小鼠视网膜血管系统的形成及维持正常生理功能可能至关重要。     

Intraretinal oxygen consumption in the rat in vivo

PURPOSE: To make the first quantitative assessment of the rate of oxygen consumption in high oxygen-consuming layers of both the outer and inner retina of the rat in vivo. METHODS: Oxygen-sensitive microelectrodes were used to measure the oxygen tension as a function of depth through the retina in anesthetized rats. Individual PO2 profiles were fitted to a multilayer mathematical model of PO2 distribution that is able to determine the oxygen uptake in those retinal layers in which the oxygen supply is through diffusion from vascular layers of the retina. This includes the entire outer retina and the region of the inner retina containing the inner plexiform layer. Measurements were performed in the light (13 mW/cm2 at the cornea) and in the dark and the amplitude and time constant for light-induced changes in outer retinal oxygenation determined. RESULTS: Under light-adapted conditions, the oxygen consumption of the outer retina was 148 +/- 11 nL O2/min/cm2 (n = 20) [corrected] and that of the included portion of the inner retina was 184 +/- 17 nL O2/min/cm2 [corrected]. In the dark, outer retinal oxygen consumption increased by 47.8% (P < 0.001), and the time constant for the resultant PO2 decrease in the outer retina was 14.9 +/- 1.8 seconds (n = 16). There was no significant change in inner retinal oxygen consumption between light and dark conditions (P = 0.89). CONCLUSIONS: Under light-adapted conditions the oxygen uptake by the selected region of the inner retina (primarily the inner plexiform layer) is greater than that of the outer retina (P < 0.01). Dark adaptation rapidly and significantly increases outer retinal oxygen consumption, but the inner retina remains unaffected.     

高度近视眼中心凹脱离伴视网膜劈裂的形态学观察

目的观察高度近视眼中心凹脱离伴视网膜劈裂的形态学特征,并探讨高度近视眼玻璃体、黄斑及后巩膜葡萄肿三者间的关系。设计回顾性病例系列。研究对象29例(38眼)高度近视眼中心凹脱离伴视网膜劈裂的患者。方法所有患者均进行裂隙灯前置镜、三面镜、直接或间接检眼镜、B超及相干光断层扫描(OCT)检查,观察黄斑中心凹脱离及视网膜劈裂的形态及其与玻璃体以及后巩膜葡萄肿的关系。其中10眼行玻璃体手术。主要指标形态学特征。结果裂隙灯前置镜、三面镜检查见视网膜成微囊样改变并浅脱离,未见黄斑裂孔。B超显示后极部视网膜水肿或浅脱离0.5~2.0mm。所有38眼的OCT扫描均显示黄斑区神经上皮脱离;未显示黄斑裂孔;神经上皮层劈裂,表现为内层劈裂、外层劈裂和双层劈裂,劈裂的内外层视网膜之间可见桥柱样连接;3眼黄斑区未见劈裂,扫描至后巩膜葡萄肿边缘附近见视网膜劈裂;视网膜前存在低反射细光带(玻璃体后皮质),且与视网膜间存在点、线及片状粘连,相应粘连处视网膜被牵引。10眼玻璃体手术中见玻璃体液化、不完全后脱离,后极部玻璃体后皮质与视网膜粘连紧密,尽量剥除后皮质,术后视网膜均复位。结论OCT可清晰显示高度近视眼中心凹脱离伴视网膜劈裂的形态特征,来自玻璃体皮质的牵引及后巩膜葡萄肿因素是导致视网膜劈裂的主要原因。     

Alterations in the differentiation of chick retina caused by an intraocular injection of an alkaline phosphatase inhibitor

Intense alkaline phosphatase (ALPase) activity has been localized in the outer plexiform layer of the developing chick retina. To elucidate the functional significance of this enzymatic activity, we have injected an ALPase inhibitor, levamisole, into embryonic eyes on either the 13th or 15th day of incubation. The retina was fixed between the 15th and 20th day of incubation and examined by electron microscopy. Levamisole injection on the 13th day caused various morphological alterations in retinal development, including the appearance of solitary photoreceptor cells in the subretinal space as well as folding of both the outer plexiform and outer nuclear layers. Pedicles of photoreceptor cells in the outer plexiform layer displayed rather smooth configurations with a reduced number of invaginations by post-synaptic neurites. The outer plexiform layer was thinned and the neuritic extensions in this layer appeared much less developed than in the control (PBS-injected) retina. Photoreceptor outer segments were seldom observed. Besides these alterations, layers of optic fibers and ganglion cells were also affected, as shown by evidence of degeneration in the ganglion cells and thinning of the nerve-fiber layer. Injection of levamisole into day 15 embryonic eyes exerted less influence on retinal development, but some photoreceptor cells were still found in the subretinal space. Some of these observations have been reported in the retinas of aged normal animals or in retinas with hereditary or induced retinal dystrophy. It is suggested that ALPase activity in the outer plexiform layer of the developing chick retina may be important for the onset of normal development of synapses in the outer plexiform layer and differentiation of the photoreceptor cells.     

婴儿黄斑区视网膜发育变化的动态观察

目的 观察婴儿视网膜黄斑区形态及视网膜各层厚度变化。方法 随机选取新生儿重症监护病房中的婴儿58例86只眼纳入研究。根据不同矫正胎龄对婴儿进行分组。其中,矫正胎龄＜32周组10例14只眼;33～36周组26例39只眼;37～41周组12例18只眼;≥42周组10例15只眼。随机选择无器质性眼病的成人12名22只眼作为成人组。所有婴儿及成人均行便携式光相干断层扫描（OCT）检查。着重观察婴儿黄斑区形态。同时,测量婴儿及成人黄斑中心凹及距中心凹750、1500 μm处旁中心凹的神经上皮层、内层视网膜、外层视网膜、神经纤维层、节细胞层、内丛状层、内核层、外丛状层、外核层厚度。分析视网膜各层厚度与矫正胎龄的相关性。结果 婴儿早期的黄斑中心凹凹陷浅,随着矫正胎龄增长凹陷逐渐加深并接近成人形态。婴儿外层视网膜结构较内层完善,随着矫正胎龄的增长逐渐出现外界膜、光感受器内外节连接（IS/OS）、光感受器外节/视网膜色素上皮（OS/RPE）层。外界膜、IS/OS、OS/RPE层最早出现时间分别为矫正胎龄32+6、35、47+6周。RPE层、脉络膜血管层厚度均随矫正胎龄增长逐渐增厚。婴儿组、成人组之间距中心凹750 μm处内层视网膜、1500 μm处神经纤维层和中心凹及距中心凹750、1500 μm处节细胞层厚度比较,差异无统计学意义（P＞0.01)。其他视网膜各层厚度在婴儿组及成人组间,以及在不同矫正胎龄组组内间差异均有统计学意义（P＜0.01）。相关性分析发现,除节细胞层外,其他视网膜各层厚度均与矫正胎龄有相关性(P＜0.05)。结论 婴儿早期的黄斑中心凹凹陷浅,随着矫正胎龄增长凹陷逐渐加深并接近成人形态。婴儿早期黄斑区外层视网膜逐渐增厚,其中以内外核层增减变化最为明显。视网膜各层随着矫正胎龄增长逐渐增厚,但发育速度并不完全一致。