酶分层消化法获取视网膜纯感光细胞

目的：探讨分离视网膜感光细胞的方法。方法：采用0．25％胰酶（视网膜感光细胞面朝下法）和2．5％胰酶＋0．2％胶原酶＋0．4％EDTA的消化液（视网膜感光细胞面朝上法）酶分层消化法分离获取纯视网膜感光细胞。结果：酶分层消化法可分离获取纯视网膜感光细胞。结论：酶分层消化法是一种可靠、费用廉价的获取纯视网膜感光细胞的新方法。     

准分子激光切削视网膜制备感光细胞片层时存在的一些问题

目的：分析准分子激光切削视网膜制备感光细胞片层时存在的一些问题。方法：以新生小牛视网膜为研究对象,白明胶作载体,行视网膜预定位,液态视网膜膜片法辅片,准分子激光PTK切削方式。结果：发现准分子激光切削时,可以产生感光细胞片层的“锁边”现象;外颗粒层细胞间的离散现象;感光细胞层内细胞核的丢失;同一切削深度下不同植片切削深度不一和同一植片内切削浓度不均等现象。结论：准分子激光切削视网膜时存在的各种问题将会直接影响视网膜移植的手术效果,必须心早解决。     

准分子激光80μm深度切削制备感光细胞片层

目的 探讨PTK切削方式下，准分子激光切削视网膜制备理想感光细胞片层所需的最佳切削深度。方法 以新生小牛视网膜为研究对象，白明胶作载体，液态视网膜铺片法铺片，准分子激光PTK切削方式，共切削3种深度，制备48个感光细胞片层，行光镜和电镜观察。结果 用准分子激光PTK切削方式，80μm切削深度下可以获得较好的视网膜感光细胞片层，光镜和扫描电镜均显示完整的感光细胞节段、外颗粒层、外网状层以及部分残存的内颗粒层细胞，而50μm和100μm深度则分别较浅和较深。结论80μm是准分子激光PTK方式切削视网膜制备感光细胞片层的适宜切削深度。     

明胶载体用于感光细胞片层制备存在的问题

目的：介绍明胶载体用于视网膜切削和制备感光细胞片层时存在的一些问题,方法：以新生小牛视网膜为研究对象,明胶作载体,常规视网膜铺片法铺片,准分子激光PTK切削方式,H－E染色和冰冻切片分析,结果;研究发现,在制备过程中可以出现感光细胞节段脱落,感光细胞核脱落和丢失,视网膜层间和层内分离以及冰冻切片时感光细胞破坏严重等现象,结论：改良明胶载体的供体形式和物理特性是未来视网膜移植 研究中的一个重要课题。     

激光切削制作视网膜切片

目的 探索视网膜横切面切片方法，显示视网膜细胞层以利于实验观察。方法 取20个健康供体眼杯，进行视网膜铺片，应用193nm准分子激光治疗性角膜切削（PTK）模式，采取不同的切削深度切削视网膜，分别用光学相干断层扫描（OCT）及苏木精-伊红染色观察剩余视网膜厚度及形态，分析切削深度与切削参数之间的关系。结果 OCT测量结果发现铺片切削深度参数与OCT测量值之间差异较大；两者之间呈正相关。石蜡切片观察见感光细胞向上切削60～90μm时，所有铺片均残余部分感光细胞内节；神经节细胞向上切削40μm时，所有铺片均残余部分神经纤维层，切削160μm时，剩余视网膜标本结构不完整。结论 激光切削制作视网膜切片操作简单，行之有效。     

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。     

准分子激光切削视网膜板层移植术

目的 应用准分子激光切削视网膜获得纯视网膜光感受器层并制作视网膜板层移植的动物模型。 方法 准分子激光切削Wistar大鼠视网膜获得纯视网膜光感受器层,植于成年RCS(Royal College of Surgeons)大鼠视网膜下，术后1个月取材，切片，光镜下观察。 结果 准分子激光切削视网膜可获得均匀整齐的视网膜光感受器层，移植后1个月，光感受器层成活对位良好。 结论 准分子激光切削视网膜光感受器板层移植可获得具有较多移植细胞，并有良好解剖学对位的视网膜移植动物模型。 (中华眼底病杂志,1998,14:209-211)     

视网膜定位联合液相铺片法用于感光细胞片层制备研究

制备高质量的感光细胞片层是取得良好视网膜移植手术效果的前提[1] 。为此 ,应首先做到以下几点 :(1)在准分子激光切削时 ,视网膜铺片必须平整 ;(2 )各视网膜植片切削深度应均一 ,即各植片在细胞成分构成上相同或接近 ;(3)激光切削要彻底 ,即整个感光细胞片层均能被全部切削。要达到以上要求 ,必须建立一套完整的视网膜定位、取材及铺片操作程序。笔者自行设计的视网膜定位联合液相铺片法技术 ,经实际应用效果满意。材料与方法一、材料1 视网膜来源 :采用北京市红星屠宰场新出生小牛视网膜。摘取小牛眼球置于冷生理盐水中保存 ,4ｈ内完成眼…     

分离视细胞层的新技术

目的为了进行视网膜视细胞移植研究，必须分离出可供移植的视细胞。能否得到纯视细胞，是对视网膜视细胞移植实验获得正确分析和良好结果的关键。方法采用一种从视网膜中分离切取纯视网膜视细胞层的可靠方法。用组织细胞震动切削机（ｖｉｂｒａｔｏｍｅ）切削视网膜，取得纯视网膜视细胞层。结果得到了可供移植的小鼠、大鼠、兔、猴及人的纯视网膜视细胞层。结论用此方法可获得视细胞纯度达１００％的纯视细胞层，方法可靠，操作较为简便。     

视细胞移植术后RCS鼠视网膜谷氨酸的分布

目的 通过纯视细胞移植,观察重建视网膜神经传导通路中兴奋性神经递质谷氨酸的分布和变化。方法取同龄Wistar鼠和皇家外科学院鼠(RCS)为供／受体,准分子激光切削法制备视细胞层,外路途径移入RCS鼠的视网膜下腔,术后两周取RCS鼠手术眼,手术对照眼,RCS对照眼,Wistar鼠眼。分别做冰冻切片,免疫组织化学染色法染色,普通光学显微镜下观察。结果在视细胞移植术后两周,移植视细胞存活,外丛状层重建,与手术对照眼、RCS对照眼相比,受体RCS鼠重建视网膜中在内丛状层和节细胞层Wistar鼠谷氨酸免疫反应阳性的神经纤维相对密度增多。结论视细胞移植不仅可以使RCS鼠视网膜重建正常的解剖结构,而且在移植后的视网膜中观察到兴奋性神经递质谷氨酸的分布接近正常。     

视网膜视细胞的成片移植

目的 探索用准分子激光切削技术制备视网膜单层细胞植片,经内入路视网膜下腔的单层视细胞成片移植。方法 用准分子激光对大鼠视网膜进行切削,制取单层视细胞植片,此后,按内入路手术方法进行了兔视网膜下腔的异种移植。结果 切削后所得视细胞植片由单层视细胞组成,结构完整,包括外丛状层、外核层和外节层;视细胞植片经明胶包埋后被准确植入宿主视网膜下腔中,移植术后第1,2天宿主观视网膜未能复位,呈脱离状态,移植物没能与视网膜色素上皮层相贴;移植后10天,宿主视网膜复位,视细胞移植片平铺于宿主视网膜下腔中,植片视细胞外节也宿主视网膜色素上皮层相贴;移植后10天,宿主视网膜复位,视细胞移植片平铺于宿主视网膜下腔中,植片视细胞外节与宿主视网膜色素上皮层相贴,未见明显免疫排异现象。结论 准分子激光制备单层视细胞植片方法简单、可行;初步观察到内入路单层视细胞成片移植后,视细胞植片能够在宿主视网膜下腔中以正常生理位置存活;视网膜下腔为理想的视网膜移植的受位。     

鼠视网膜感光细胞移植术后超微结构观察

目的 以遗传性视细胞变性动物ＲＣＳ鼠为受体,Ｗｉｓｔａｒ鼠为供体,观察纯视锥、杆细胞移植术后ＲＣＳ鼠视网膜外层超微结构的变化。方法 应用视网膜板层切削技术或准分子激光技术制备供体层状视细胞;视网膜板层外路移植技术,获得视网膜移植动物模型。术后２周及４周时处死动物,眼球常规切片后于光镜及透射电镜下观察受体视网膜。结果 移植的视细胞大多成层排列于受体鼠的视网膜色素上皮层及内颗粒层之间。重新出现的外网状     

快速退变性视网膜变性感光细胞超微结构变化分析

目的 了解快速退变性遗传性视网膜变性的感光细胞在出生后早期发生的形态学变化,为临床治疗提供依据.方法 取出生后不同时间的rd小鼠及正常对照小鼠各6只的视网膜,经光镜、扫描电镜和透射电镜观察感光细胞的形态发生发育和结构变化过程,比较二者的动态变化和形态学差异.结果 rd鼠生后1周开始出现感光细胞节段变短,内节段内线粒体变性改变多见;偶见感光细胞核旁胞浆内出现肿胀变形的线粒体.2周时节段层变薄,内节段结构已不完整,外节膜盘少见,变形且排列不整齐;外核层细胞层数明显减少,可见核固缩及染色质凝聚,偶见外丛状层部分神经突起内出现变性的线粒体.3周时节段层近消失,外核层只剩一层细胞,胞浆内可见髓样结构;外丛状层变薄.4周时内节段高度变形,视网膜色素上皮层与外核层之间出现大量不成形结构.外核层仅残存少许胞体,胞浆内出现多量不成形结构.外丛状层极薄,部分区域已消失.结论 rd鼠在出生后早期就发生感光细胞的变性改变,细胞内线粒体改变明显.其感光细胞变性发生早,进展快,呈快速退变特点.     

Delayed retinal effects of the frequency-doubled YAG laser (532 nm)

In order to compare the retinal effects of the frequency-doubled YAG laser (532 nm) with those of argon laser, rabbit eyes were exposed to green YAG laser irradiation and processed for light and electron microscopic study at 24 hr, 2 weeks and 4 weeks. Detailed analysis was conducted on tissue exposed to 7.3 and 7.6 millijoules (mj). Response of the photoreceptors and retinal pigmented epithelium to green YAG was very similar to that described for argon laser over the same time period. By 2 weeks post-exposure, there was histologic evidence of partial recovery with absence of damaged, pycnotic photoreceptor nuclei, increased numbers of typical photoreceptor outer segment lamellae and repair of retinal pigmented epithelium. Four weeks after irradiation, normal-appearing photoreceptor nuclei were present although inner photoreceptor segments still showed mitochondrial damage. Outer segments at 4 weeks showed regular lamellar structures. We conclude that the frequency-doubled YAG laser is equivalent to the argon laser with respect to the production of thermal lesions in the retina. Its additional advantages include increased efficiency, portability, reliability and lack of absorption by macular xanthophyll pigment.     

Cones regenerate from retinal stem cells sequestered in the inner nuclear layer of adult goldfish retina

PURPOSE: To determine whether retinal progenitor cells in the inner nuclear layer give rise to regenerated cones after laser ablation of photoreceptors in adult goldfish retina. METHODS: Using a technique developed previously in this laboratory, photoreceptors in the retina of adult goldfish were ablated with an argon laser. The mitotic marker, bromodeoxyuridine, was used to label proliferating and regenerated cells, which were identified with cell-specific markers. RESULTS: Cells proliferating locally within lesion included microglia, Müller glia, and retinal progenitors in the inner nuclear layer (INL). The nuclei of both Müller glia and associated retinal progenitors migrated from the inner to the outer nuclear layer. The proliferating retinal progenitors, which express Notch-3 and N-cadherin, regenerated cone photoreceptors and then rod photoreceptors. CONCLUSIONS: Previous work has demonstrated that photoreceptors in the goldfish retina regenerate selectively after laser ablation, but the source of regenerated cones has not been identified. The results reported here provide support for the existence of retinal stem cells within the adult fish retina that are capable of regenerating cone photoreceptors. The data also support the involvement of Müller glia in the production of regenerated cones.     

Ultrastructural localization of retinal S-antigen in the rat

Using a peroxidase-antiperoxidase immunohistochemical technique, the light and electron microscopical localization of retinal S-antigen was demonstrated in the rat retina, using antisera to bovine and guinea pig S-antigen. Comparable localization of S-antigen was observed with both antisera, although in our hands, the antiserum to bovine S-antigen gave more intense immunostaining.By light microscopy, the most intense immunoreactivity to retinal S-antigen was seen in the outer segment of the photoreceptor layer. Some immunostaining was also demonstrated surrounding the cell bodies of the outer nuclear layer. The inner segment of photoreceptors was virtually devoid of immunoreactivity. Ultrastructurally, immunoreactivity to S-antigen was demonstrated mainly on the inner surfaces of the visual discs of the rods. The plasma membranes of the outer segment of the rods were stained as well. Immunoreactivity surrounding the cell bodies of the outer nuclear layer was ultrastructurally localized on plasma membranes. The inner segment of the photoreceptor layer, other parts of the retina, or any eye structures other than retina showed no immunoreactivity.The relationship of retinal S-antigen to other cell components in the retina and the possible mechanisms of the autoimmune uveal diseases are discussed.Previously published in a short form at the ARVO meeting in 1983