对鸡胚视网膜细胞DNA合成的放射自显影研究

目的　研究鸡胚视网膜细胞发生过程中的形态变化与DNA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鸡胚视网膜氚标胸腺嘧啶的分布进行观察。 结果　随胚龄增加氚标胸腺嘧啶的银颗粒数,在视杯后部由多到少,而在视杯前部则由少到多,均以 2d胚达高峰。胚龄早期各部银颗粒数外层多于内层;但胚龄晚期则内层增多,外层消失。 结论　视网膜细胞的分化成熟由后部移向前部,视细胞和视神经细胞分化较早;伴随视网膜细胞的分化成熟,DNA的合成数量和区域分布表现为动态变化。     

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)     

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，     

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.     

一种稳定标记小鼠视网膜内两种不同类型神经节细胞的方法

目的 报道一种简单高效显示小鼠视网膜中两种不同类型神经节细胞的方法.方法 利用特殊标记物Brn3a和Melanopsin,通过视网膜铺片免疫荧光双标染色结合激光共聚焦显微镜,分别标记小鼠视网膜中普通视网膜神经节细胞和内在光敏视网膜神经节细胞.结果 免疫荧光染色结果表明,内在光敏视网膜神经节细胞与普通视网膜神经节细胞均位于视网膜节细胞层,相间互补分布.内在光敏视网膜神经节细胞数量较少,为普通视网膜神经节细胞的1％ ～2％,其轴突朝向视神经盘方向汇集,树突野较大,伸向内网层.结论 免疫荧光双标染色是小鼠视网膜内两种不同类型视网膜神经节细胞简单易行、稳定高效的标记方法.     

大鼠骨髓间充质干细胞视网膜下移植的短期观察

目的探索骨髓间充质干细胞（MSCs）视网膜移植的可行性。方法采用贴壁筛选法分离、培养SD大鼠骨髓MSCs，将溴脱氧尿嘧啶（BrdU）标记的大鼠MSCs悬液经玻璃体腔注入大鼠视网膜下腔。术后14d处死大鼠，取新鲜眼球，连续冰冻切片，经BrdU单抗、FITC标记二抗和Rhodopsin单抗、Cy3标记二抗免疫荧光双重标记后，荧光显微镜下观察。结果荧光显微镜下可见移植后的MSCs主要分布于视网膜色素上皮层和视锥、视杆细胞层，未表达视网膜光感受器细胞的特异性抗原视紫红质。结论移植14d后MSCs可在视网膜下腔存活，主要分布于视网膜色素上皮层和视锥、视杆细胞层，未分化成视网膜光感受器细胞。     

视网膜对铁的敏感性与抗坏血酸分布的相关性研究

目的 观察大鼠和家兔视网膜组织中抗坏血酸的分布形式与铁异物对2种不同动物的视网膜损伤形态,验证大鼠视网膜光感受器细胞对铁异物的敏感性是否与视网膜中抗坏血酸的分布有关。 方法 将5 mg铁异物分别置入32只健康雄性Sprague-Dawley (SD ) 大鼠、15 mg铁异物分别置入9只健康雄性新西兰白色家兔玻璃体腔后，苏木精-伊红（hematoxylin-eosin, HE）染色观察2种不同种动物视网膜的受损情况。凋亡原位染色（TdT-mediated dUTP-biotin nick-end labeling, TUNEL）方法观察家兔视网膜神经元的细胞凋亡。Chinoy 法染色观察抗坏血酸在2种不同的正常动物视网膜组织中的分布形式。 结果 SD大鼠铁异物置入术后，HE染色的切片仅见光感受器细胞层的破坏。新西兰白色家兔铁异物置入术后3 d，HE 染色见全层视网膜神经元细胞受损，在神经节细胞层、内颗粒层以及外颗粒层均观察到TUNEL阳性细胞核。正常SD大鼠抗坏血酸阳性染色仅见于外颗粒层；新西兰家兔视网膜全层抗坏血酸染色阳性。 结论 视网膜对铁异物毒性的敏感性与视网膜组织中的抗坏血酸分布形式有关。 （中华眼底病杂志，2003，19：269-332）     

成年大鼠视网膜中脑红蛋白表达的亚细胞分布

目的:电镜下观察正常成年大鼠视网膜中脑红蛋白(Ngb)的表达。方法:用电镜免疫金-银细胞化学法研究Ngb在成年大鼠视网膜中的亚细胞定位。结果:Ngb在视网膜除外核层和视锥视杆层的外节段以外的其它各层均有分布。在细胞层Ngb定位于细胞的胞质内,线粒体周围多见,内质网和高尔基复合体的管腔外侧也可见Ngb阳性着色;在丛状层Ngb分布于突触前成分的突触小泡之间。结论:Ngb的特殊亚细胞分布提示其与神经元供氧的紧密关系。     

人胚眼视网膜光感受器细胞的体外培养和鉴定

目的：探讨人胚眼视网膜光感受器细胞的获得和体外培养方法,为进行人类视网膜光感受器细胞移植提供理论依据。方法：采用酶分层消化法分离获取了胚眼纯视网膜光感受器细胞,使用视网膜色素上细胞的条件培养液,在体外长期培养并进行形态学鉴别;用苏木素－伊红染色显示消化后的视网膜组织结构层次,以确定所获得取的视网膜光感受器细胞的纯度;用抗视杆细胞特异性视紫质蛋白抗体（rhodopsin4D2,Rho4D2)免疫细胞化学染色做细胞鉴定,结果：酶分层消化法能获取较纯的视网膜光感受器细胞,抗Rho4D2阳性,并能在体外存活较长时间,结论：酶分层消化法所获取的人胚眼纯视网膜光感受感器细胞能在体外较长时间存活,并可表达光感受器细胞特异性视紫质蛋白,将为人类视网膜光感受器细胞的移植和各种体外研究提供细胞特异性视紫质蛋白,将为人类视网膜光感受器细胞的移植和各种体外研究提供细胞来源。     

环状RNA与眼底增生性疾病的研究进展

眼底增生性疾病, 如脉络膜新生血管、糖尿病视网膜病变、增生性玻璃体视网膜病变等, 是以细胞增生或新生血管形成为病理特征, 影响眼部正常结构及功能的一组疾病。环状RNA是一种非编码RNA, 并被证实参与多种眼科疾病的病理生理过程。因此, 环状RNA有希望成为治疗眼底增生性疾病的潜在靶点之一。本文就环状RNA的生理功能及其在糖尿病视网膜病变、增生性玻璃体视网膜病变、青光眼中神经胶质细胞增生等疾病和病理过程中的作用进行综述。     

Metabolic Studies on Retinal Tissue from a Donor with a Dominantly Inherited Chorioretinal Degeneratlon Resembling Sectoral Retinitis Pigmentosa

Protein synthesis, glycosylation, RNA synthesis, and neurotransmitter uptake were monitored using biochemical and autoradiographic techniques following in vitro labeling of retinal tissue from a 79-year-old female with sectoral retinitis pigmentosa (RP). Comparisons were made between degenerate and non-degenerate regions of the RP retina, and normal retinal tissues from an age- and postmortem-matched donor. Autoradiographs of non-degenerate retina from the RP eye following ³>H-uridine incubation revealed virtually identical silver grain density over nuclei in all retinal strata as compared to normal control retinas. In contrast, a photoreceptor-specific reduction in silver grain density in the non-degenerate RP retina was noted following ³H-leucine incubation. In the normal retina, rod photoreceptor labeling with ³H-mannose was always greater than cone photoreceptor labeling. This pattern of incorporation was reversed in the non-degenerate region of the RP retina where rod photoreceptor labeling was less pronounced than that observed for cone photoreceptors.In non-degenerate regions of the RP retina, a marked accumulation of ³H-GABA by the M?ller's cells was observed. Few cells exhibited selective uptake of³>H-muscimol, a GABA analog, indicating that few GABAergic neurons remained in the degenerate retina. ³H-dopamine-accumulating cell terminals were observed in the usual positions in the non-degenerate RP retina. In the degenerate region of the RP retina, heavy and diffuse uptake of ³H-GABA and³>H-muscimol, respectively, into broad cellular processes were noted, whereas³>H-dopamine was accumulated by only a few punctate terminals.     

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.     

Effects of monosodium glutamate on chick embryo retina in culture.

Monosodium glutamate added to 12-day chick embryo retinas in culture causes severe morphologic damage to the retina as judged by light microscopic examination. Damage is evident after a few hours with concentrations as low as 0.3 mM. Glutamyltransferase induction is also appreciably inhibited by the amino acid. General protein synthesis and RNA synthesis appear to be less affected.     

Biogenesis and content of rhodopsin in the retina of the chick during development

Developmental aspects of the formation of rhodopsin in the chick were investigated. The content of rhodopsin was measured in the retina of the developing chick embryo, newly hatched and adult chickens by both spectral and immunological procedures. Rhodopsin was first detected spectrally at day 18 (stage 43) with the level increasing about 6 fold through hatching. The concentration in the adult retina was about one half that present in the 1 day old chick. By the more sensitive procedure of radioimmunoassay (RIA), rhodopsin was detected as early as day 12 (stage 37), but was undetectable in the 8 day old embryo. The rhodopsin concentration remained low and relatively constant until day 17 (stage 42), at which point it increased rapidly up to hatching. Retina from the newly-hatched chick had a rhodopsin concentration some 30 fold higher than the 16 day old embryo (stage 41). The results of RIA indicate that the biosynthesis of rhodopsin may significantly precede the morphological appearance of outer segments.     

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.     

Cholinergic neurons in the human retina

Choline acetyltransferase (ChAT)-like immunoreactivity in the human retina can be demonstrated using a polyclonal antiserum to ChAT isolated from chick brain. There is a population of ChAT-like immunoreactive cells along both margins of the inner plexiform layer (IPL). The labeled cells have a morphology and position characteristic of the cholinergic amacrine- and displaced amacrine cells demonstrated by other workers in the mammalian retina. Non-immune rabbit serum or pre-absorbed antiserum, used in place of the primary antiserum, verified the specificity of the method. Human retinas can also be labeled with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI), which has been reported to bind selectively to DNA in the nuclei of cholinergic cells. The fluorescent cells are similar in morphology, position, and distribution to the cells which show ChAT-like immunoreactivity. In addition, we have localized the presence of [3H]choline and [3H]choline metabolites in freeze-dried, vapor-fixed tissue using 'dry' autoradiographic techniques. Incubation in [3H]choline was followed by either stimulation or inhibition of calcium-dependent transmitter release during a 1-hr 'chase' period. Using tissue incubated in a chase designed to retain labeled neurotransmitters, silver grains were concentrated over a population of cell bodies at either margin of the IPL (i.e. in the same position as putative ChAT-immunoreactive cells and DAPI-labeled cells). In contrast, tissue incubated in a chase designed to release labeled acetylcholine was labeled uniformly throughout the neural retina, with a heavy band of label over the pigment epithelium. Taken together, the results presented here indicate that three independent markers for cholinergic cells (i.e. ChAT immunoreactivity, DAPI binding, and choline uptake) are present in a population of cells in the human retina. This suggests that acetylcholine may be a neurotransmitter synthesized by amacrine and displaced amacrine cells in the retina.     

Cellular abnormalities of fast-growing chickens: Similarities between retina and lens

A comparison of cell behaviour in culture conditions of embryo chick neural retina cells from Normal and Hy-1 genotypes shows that the Hy-1 cells exhibit anomalies in behaviour similar to those previously demonstrated for Hy-1 lens cells, including reduced plating, high cell-cell aggregation and mitotic rate.     

Uptake of (3H)cortisol by chick embryo retina as a function of age

[³H]Cortisol is rapidly taken up by chick embryo retinas placed in culture medium containing the steroid. The accumulation of cortisol within the retina increases with time up to about 30 min. Retinas used were from embryos ranging from 7 to 17 days of development. Although retinas from younger embryos are relatively refractory to induction by cortisol, accumulation of the steriod at equilibrium is higher in the younger tissues than in the older, more inducible ones.