兔视网膜色素上皮细胞移植的实验研究

目的：探索用改良的二步酶法制备视网膜色素上皮（RPE）细胞悬液，经改良的内路法，不经玻璃体切割的情况下进行视网膜下间隙的移植，方法：用酶1（含220kU/L透明质酸酶和0．1％的胰蛋白酶）松解视网膜神经上皮细胞与RPE细胞之间的联接，再用酶II（含0．25％胰蛋白酶）从Bruch膜上离散RPE细胞，制成细胞悬液，用特制的注射针头，通过人为造成的局限性视网膜脱离区将其移植入受体视网膜下间隙，结果：术后2周供体RPE细胞层呈单层排列于受体视网膜下间隙，并存活，未见明显炎症反应，电镜观察，术后7周供体RPE细胞位于受体Bruch膜上并与受体感光细胞外节盘模型成镶嵌关系。结论：改良的内路法可以将供体RPE细胞成功移植在受体眼视网膜下间隙，并至少存活7周，改良的内路法渴望为视网膜移植的实验室研究提供一条可供借鉴的途径。     

兔眼虹膜和视网膜色素上皮细胞移植的形态学观察

目的 比较有色兔虹膜色素上皮及视网膜色素上皮细胞在白兔视网膜下腔的生长状况。 方法 分离培养有色兔的IPE及RPE细胞。采用内路法在8只白兔16眼中进行了IPE和RPE细胞悬液的移植,其中左眼移植IPE细胞,右眼移植来自同一供体兔的RPE细胞。8只兔分别在2周(n=3)、4周(n=3)和6周(n=2)时处死。对眼球壁做光镜和电镜检查。 结果 IPE和RPE细胞在异体视网膜下腔存活,绝大多数贴附在Bruch’s膜上,并具有极性,细胞基底部有大量的皱褶,细胞顶部有一些微绒毛。6周时未见移植细胞周围有炎性细胞浸润。 结论 移植的IPE和RPE细胞可以在视网膜下腔存活,它们的形态均和原先RPE细胞类似。IPE细胞有望替代RPE细胞用于移植。     

兔眼虹膜色素上皮细胞自体视网膜下移植的研究

目的探讨兔眼虹膜色素上皮(IPE)细胞的自体移植技术并进行术后移植区的细胞形态学观察。方法通过虹膜全切法获取10只新西兰白兔的虹膜,采用酶-机械分离-酶消化的方法分离IPE细胞,用5溴脱氧尿嘧啶(5-Brdu)标记原代或一代的细胞。将标记过的IPE细胞悬液通过内路法分别植入对侧眼的视网膜下腔。术后2、4、6周摘除眼球对移植区进行光镜、电镜检查。结果经免疫学鉴定纯化培养了兔眼IPE细胞。术后光镜显示移植的IPE细胞成单层贴附在原始的视网膜色素上皮(RPE)细胞上。电镜结果证实移植的IPE细胞尖端微绒毛与光感受器外节连接,邻近的光感受器细胞显示正常结构。结论采用内路法可成功地将细胞悬液植入家兔视网膜下腔。6周内移植的自体IPE细胞在视网膜下腔存活,与原先的RPE细胞生长状态类似,邻近的光感受器细胞无形态改变。     

视网膜色素上皮移植方法的探讨

目的；探讨视网膜色素上皮移植的方法。 方法：以常规方法割备有色素兔眼视网膜色素上皮(RPE)细胞作为供体，移植到10只无色素兔眼．8只眼经外路法，即穿过巩膜和脉胳膜的方法；另10只眼内路法，即从睫状体平部进入玻璃体，制成人为的局限性视网膜脱离，将RPE细胞移植到受体眼视网膜下腔。术后第10、20、40、90天作光镜和电镜检查。 结果：内路法组，手术后40天光镜观察，移植区和非移植区神经视网膜层厚度无差别．透射电镜显示：移植的RPE细胞嵌入Bruch膜，光感受器外节在正常位置，术后90天，移植细胞内发现吞噬外节的次级溶酶体．外路法的8只眼因脉胳膜出血或视网膜穿破而失败。 结论：内路法是RPE细胞移植切实可行的方法。 （中华眼底病杂志，1997，13：160-162）     

视网膜色素上皮移植细胞超微结构的观察

目的 观察视网膜色素上皮移植细胞的超微结构,为临床应用提供实验基础。方法 取有色兔眼球后段,去除视网膜的神经上皮层,获得视网膜色素上皮细胞作为移植的供体细胞。以１０只无色兔为移植的受体,内路法将供体色素上皮细胞移植到视网膜下腔,电镜观察移植细胞的超微结构。结果 移植的１０只眼,细胞成功移植到视网膜下腔,可见移植细胞粘附于受体的Ｂｒｕｃｈ膜并形成基底内褶,细胞间紧密连接,细胞内可见吞噬体。结论 内路     

玻璃体内注射他克莫司对异种移植的视网膜色素上皮细胞存活的影响

目的 观察少量、多次玻璃体内注射免疫抑制剂他克莫司（FK506）对移植到兔眼视网膜下间隙内的人视网膜色素上皮（retinal pigment epithelium, RPE）细胞存活情况的影响。 方法 采用绿色荧光蛋白（green fluorescent protein, GFP）逆转录病毒感 染人RPE细胞。将50 μl（4×10³个/μl）表达GFP的人RPE细胞悬液注射到18只白兔及1 0只灰兔双眼视网膜下间隙，手术后所有兔左眼玻璃体内注射5 μl FK506（5 μg/ μl），每周1次，连续5周，然后间周一次至20周;右眼不注射作为对照。眼球壁铺片倒置荧光 显微镜观察移植细胞的存活状态。 结果 白兔于移植后1、2、3、4 、6、10、11、14、18、20、23、24、25、33、54周，灰兔于移植后4、5、6、7、14、18、20、26周双眼视网膜下均可见表达的GFP细胞，但移植术后1~14周内玻璃体内注射了FK506的左眼视网膜下RPE-GFP细胞形态、细胞膜的完整程度均比未注射的右眼好。18周后，7只白兔和3只灰兔双眼视网膜下移植细胞的状态差异不明显。眼球切片苏木素-伊红（hematoxylin-eosin, HE）染色观察结果显示，移植术后1~6周，6只白兔和3只灰兔右眼脉络膜小血管周围可见灶性或弥散的淋巴细胞，应用了FK506的左眼淋巴细胞的浸润明显减少。 结论 移植术后早期玻璃体内使用小剂量免疫抑制剂可减轻局部炎症反应，有利于维持视网膜下间隙移植的异种细胞的存活。 （中华眼底病杂志，2003，19：333-404）     

激光光凝后视网膜色素上皮、Bruch膜和脉络膜超微结构改变

目的观察激光光凝后视网膜色素上皮(RPE)、Bruch膜和脉络膜超微结构的改变和修复过程。方法收集眼眶恶性肿瘤患者7例,其眼球的视网膜及脉络膜结构基本正常。均经患者签署知情同意书后,于眶内容物剜除术前1、3、7d,用Ⅰ、Ⅱ、Ⅲ级光斑光凝视网膜。摘除眼球后,将其视网膜和脉络膜组织制成超薄切片、醋酸铀和枸橼酸铅染色,于透射电镜下观察其超微结构。结果激光光凝后1d,经Ⅰ级光斑光凝视网膜组织见部分RPE细胞肿胀、坏死、数量减少,微绒毛部分消失;Bruch膜完整;脉络膜毛细血管内皮肿胀,少数管腔闭塞。经Ⅱ级光斑光凝的视网膜组织见较多RPE细胞溶解破坏,Bruch膜空泡形成,脉络膜毛细血管广泛闭塞。经Ⅲ级光斑光凝的视网膜组织见大量RPE溶解消失,Bruch膜部分破坏,脉络膜血管受损。激光光凝后3d,RPE细胞和脉络膜水肿减退,破坏区内出现吞噬细胞,RPE细胞和脉络膜纤维组织开始增生。激光光凝后7d,增生的RPE细胞覆盖破坏区,脉络膜血管减少和基质纤维化。结论激光光凝可引起RPE细胞水肿、坏死,Bruch膜和脉络膜血管不同程度破坏,随后RPE细胞和纤维组织增生修复破坏区。不同参数的激光治疗可修复病变的RPE和脉络膜组织。（中华眼科杂志,2004,40：692-695)     

视网膜色素上皮细胞移植的研究

视网膜色素上皮 (retinal pigm ent epithelium,RPE)由排列规则的单层细胞构成 ,位于视网膜神经上皮层和 Bruch膜之间 ,由于其特殊的解剖学位置 ,它在视网膜中起着重要的作用 [1 ]。 RPE、Bruch膜和脉络膜毛细血管的进行性病变可危及视网膜感光细胞 ,形成视网膜下的新生血管膜 ,比如老年黄斑变性 (age- related macular degeneration,ARMD)。传统的光凝疗法预后不佳 ,尤其在黄斑区 [2 ,3] 。近年来成功开展了视网膜下膜的“剥膜”手术 ,由于同时丧失了 RPE细胞 ,要求移植以作补充。由此带来了以下新课题 :(1) RPE的取材与培养 ;(2 )…     

Cyclosporine玻璃体腔内注射对异种视网膜色素上皮移植的排异抑制作用

目的 观察兔眼视网膜下腔植入人胚眼视网膜色素上皮（retinal pigment epithe-lium,RPE）后不同时期的眼底和组织学改变。研究环胞菌素A（Cyclosporines,CsA）玻璃体腔内注射能否抑制人胚眼RPE在兔眼视网膜下腔中诱导的异种移植排异反应。方法 人胚眼色素上皮片和浓缩色素上皮细胞悬液植入36只兔眼的视网膜下腔,其中16眼为对照组,用于观察排异的自然转规。分别7d(8眼）和30d(8眼）后获取组织标本。另20眼为实验组。RPE移植后,每周一次玻璃体腔内注射CsA 1mg(12眼）或CsA0.1mg(8眼）。视网膜和视神经的毒性反应使用ERG进行检查。结果 人胚眼RPE片和浓缩的RPE细胞均能在视网膜下腔短期存活。移植的RPE与视细胞结合良好并显示吞噬功能。排异反应发生时间约在术后10～30d。对照组中7d的排异发生率为0/8;30d排异发生率为7/8。排异发生后荧光造影中移植区为高荧光区,组织切片中显示有大量的组织细胞积聚。CsA1mg组30d排异发生率为0/12,0.1mg组为5/8。ERG波幅的下降与CsA剂量和注射次数呈正相关。结论 异种RPE视网膜下腔移植在无免疫抑制剂的条件下,只能短期存活。CsA玻璃体腔中注射能抑制异种RPE移植的排异反应但易引起明显的视网膜毒性反应。     

视网膜下间隙移植经基因修饰的视网膜色素上皮细胞对大鼠光感受器变性及视功能的保护作用

目的 以RCS大鼠作模型,研究经基因修饰的永生化视网膜色素上皮细胞(RPE)视网膜下移植对光感受器变性的保护作用。方法 在绿色荧光蛋白基因逆转录病毒感染的基础上,利用脂质体介导节状神经生长因子(CNTF)表达质粒转移,修饰成人RPE细胞系CRL-2302。将1×105个表达绿色荧光蛋白(GFP)或GFP及CNTF的RPE细胞移植到4～5周龄RCS大鼠右眼视网膜下间隙,左眼不移植或注射。PBS作为对照。术后2、4、6、8、10和12周作荧光显微镜、光镜、电镜及电生理检查。结果 荧光显微镜观察,术后1周移植的人RPE细胞在RCS大鼠视网膜下间隙已扩散到几乎整个眼底,但随时间延长移植的细胞逐渐减少,术后6周仅残留少量移植细胞。光镜及电镜观察显示移植眼保留的光感受器数量明显较对照眼多,凋亡细胞则较对照跟少。此外,移植眼宿主RPE细胞形态较正常,并可见吞噬小体。视网膜电图(ERG)检查结果表明部分移植眼视网膜功能明显较对照眼好。结论 经过基因修饰的RPE细胞移植可延缓RCS大鼠视网膜光感受器变性,为治疗视网膜变性提供了新的途径。(中华眼科杂志,2004,40:552-556)     

Transplantation of cultured rabbit retinal epithelium to rabbit retina using a closed-eye method

We have developed a closed-eye technique for transplanting cultured rabbit retinal epithelial cells to Bruch's membrane of the rabbit. A glass micropipette containing a suspension of 3H-thymidine-labeled, cultured retinal pigment epithelial (RPE) cells is inserted through a pars plana incision and positioned adjacent to the neural retina. A jet stream from the pipette is used to make a small retinal hole and bleb detachment. Patches of host retinal epithelium lift off with the neural retina, creating areas of bare Bruch's membrane. The cell suspension is injected into the subretinal space, and labeled cells can be seen attached to Bruch's membrane as early as 1 hr later. The neural retina spontaneously reattaches within 24 to 48 hr, bringing photoreceptor outer segments in direct contact with the transplanted cells. Phagocytosis of outer segment material by transplanted cells can be seen as early as 24 hr after surgery. This closed-eye technique offers an advantage over the open-sky method used previously in that it allows for reattachment of the neural retina and at least a partial return of function in the transplanted retinal epithelium.     

Vitreoretinal surgical technique for transplanting retinal pigment epithelium in rabbit retina.

Transplantation of retinal pigment epithelial (RPE) cells has been proposed as a potential remedial procedure for previously untreatable retinal diseases. In this study, a vitreoretinal surgical technique was used to transplant pigmented RPE cells obtained from pigmented rabbits into the subretinal space of New Zealand White rabbits. At the time the animals were sacrificed, the retina was re-attached in all but 4 of the 24 experimental eyes. Histologically, by one week the transplanted RPE cells had formed a monolayer in patchy areas beneath the attached retina. By electron microscopy, RPE cells with prominent melanin granules were found attached to Bruch's membrane. Three weeks after transplantation, grafted RPE cells had formed apical microvilli and tight junctions with adjacent cells. The nucleus of the cells containing pigment had become oval, and their contact with Bruch's membrane appeared to be composed of bsal infoldings that were well formed. Our findings demonstrated the functional appearance of the transplanted RPE cells.     

Identification of transplanted retinal pigment epithelium with a novel chromosomal marker

PURPOSE: To demonstrate the ability of a novel chromosomal marker to identify retinal pigment epithelium (RPE) after xenotransplantation, and determine the short-term correlation between pigment and this nuclear marker. METHODS: Primary pigmented RPE harvested from third trimester fetal pigs were transplanted as microaggregates into the subretinal space of 3 albino rabbits. We then used an in situ probe for a repetitive segment of the porcine chromosome to identify the transplanted RPE. RESULTS: Pigmented cells were visible in the subretinal space 2 weeks after transplantation. Approximately 70% of pigment-containing cells were also labeled with the porcine chromosomal marker. Labeled cells were predominantly flatter in morphology and close to Bruch's membrane whereas unlabeled cells were rounder and further from Bruch's membrane. The outer nuclear layer thickness was normal above the pigmented monolayer but was decreased over areas containing multiple layers of pigmented cells. CONCLUSIONS: Fetal porcine RPE xenografts can be identified with a nuclear marker for a repetitive segment of the porcine chromosome. The presence of pigment within unlabelled cells suggests that pigment is not a robust marker for transplanted RPE.     

Transplanted and repopulated retinal pigment epithelial cells on damaged Bruch's membrane in rabbits

AIMS—The authors studied how artificially damaged Bruch's membrane influenced growth and differentiation of transplanted embryonic retinal pigment epithelial (RPE) cells and of host RPE cells in rabbits.
METHODS—Embryonic RPE cells obtained from pigmented rabbits were transplanted into the subretinal space of adult albino rabbits. The host RPE was removed with a silicone cannula, and Bruch's membrane was damaged by scratching with a microhooked 27 gauge needle under the detached retina in closed vitrectomy. The transplantation sites were examined 3, 7, and 14 days after surgery by light and electron microscopy.
RESULTS—Varying degrees of damage in Bruch's membrane were observed. Pigmented and hypopigmented RPE cells showed a normal polarity and tight junctions were seen at the sites of mild to moderate damage 3-7 days after the surgery. In contrast, fibroblast-like cells with no such features of RPE cells formed multiple layers at the sites of severe damage involving the full thickness of Bruch's membrane and the choriocapillaris even 14 days after the surgery. Without transplantation, host RPE cells repopulated the damaged areas in the same way as transplanted RPE cells.
CONCLUSIONS—Transplanted embryonic RPE cells as well as host RPE cells grew and differentiated on the moderately damaged Bruch's membrane, while the severely damaged Bruch's membrane did not allow differentiation of RPE cells although these cells could grow and cover the damaged areas.

Keywords: transplantation; repopulation; retinal pigment epithelial cells; Bruch's membrane; rabbit     

Comparison of external and internal approaches for transplantation of autologous retinal pigment epithelium.

The feasibility of autologous transplantation of retinal pigment epithelial (RPE) cells from just posterior to the ora serrata to the posterior pole was demonstrated in the rabbit model. Two techniques for introducing the transplanted cells were compared: an internal (anterior transvitreal) and an external (posterior transscleral) penetration to the subretinal space. In both approaches, RPE cells were obtained by biopsy from the peripheral retina of a rabbit eye, cultured, labeled with a fluorescent dye and 3H-thymidine, and transplanted to the posterior pole of the same or contralateral eye. The external approach consistently resulted in a greater number of transplanted cells on Bruch's membrane. The internal technique was more precise because it permitted direct visualization of the placement of the transplanted RPE. Transplantation of autologous RPE is a possibility that should be further pursued.     

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。     

Survival of allogeneic porcine retinal pigment epithelial sheets after subretinal transplantation

PURPOSE: To examine morphology after transplantation of organized primary porcine RPE sheets into the porcine subretinal space. METHODS: Primary RPE sheets were harvested from freshly enucleated female porcine eyes and embedded in a thin slice of 50% gelatin and 300 mM sucrose before subretinal transplantation into male pigs by using vitrectomy techniques. Thirty-eight animals that underwent surgery were observed for up to 3 months without immune suppression. RESULTS: Four days after surgery, the subretinal space contained a multilayer of heavily pigmented RPE that was predominantly Barr body positive. One month after transplantation, there was marked shortening of the outer segments with an intact external limiting membrane. The transplant bed contained a pigmented monolayer in some regions, whereas in other regions the graft was folded into multilayers with degenerated inner layers of transplanted cells despite synthesis of basement membrane. The choroidal vessels and choriocapillaris remained patent in the transplant bed. Barr body positive cells were still present 3 months after surgery. There was no infiltration of the graft site with inflammatory cells. CONCLUSIONS: Allogeneic RPE grafts survive in the subretinal space up to 3 months after surgery, and the choriocapillaris remains patent in the transplant bed, although there are many heavily pigmented cells within the transplant bed that are Barr body negative by 3 months. Further work is needed to produce uniform repopulation of a sizable portion of Bruch's membrane with a monolayer of transplanted RPE.     

Survival and integration of neural retinal transplants in rd mice

PURPOSE: To examine the ultrastructure of the rd retina after transplantation of small micro-aggregates of neural retina in order to determine their survival and integration with the host retina and for sites of communication between transplant and host neurons. METHODS: Neonatal micro-aggregates from transgenic mice expressing a LacZ gene reporter gene in their rods were transplanted into the subretinal space of transgenic rd mice expressing a LacZ reporter gene in their rod bipolar cells. The mice were killed at various times after transplantation surgery and studied by light and electron microscopy. RESULTS: Retinal transplants survived well, as long as 8 months, without signs of rejection and were well integrated into the host retina. Cell bodies of transplanted rods made membrane-to-membrane contacts with rod bipolar cells of the host at areas where there were gaps in the host external plexiform layer. One synaptic process of a transplanted rod was found on the vitreal side of the host's external limiting membrane. In two cases, a postsynaptic process in a transplanted rod spherule contained an Xgal label, implying that it belonged to a host rod bipolar. There was evidence of extension of processes between host and transplant retinas involving astrocytic rather than neural structures. CONCLUSIONS: Retinal allografts to the subretinal space of rd mice survive indefinitely. Close but non-synaptic contacts occur between transplant and host neurons that could allow ephaptic communication between these two retinas. Evidence of synaptic contacts between transplant and host was difficult to find.