In vitro transplantation of fetal human retinal pigment epithelial cells onto human cadaver Bruch's membrane
In vitro transplantation of fetal human retinal pigment epithelial cells onto human cadaver Bruch's membrane
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DOI：
10.1006/exer.1997.0404
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发表时间：
1998-01-01
影响因子：
3.4
通讯作者：
Zarbin, MA
中科院分区：
文献类型：
--
作者：
Castellarin, AA;Sugino, IK;Zarbin, MA
视网膜色素上皮移植已被提出作为脉络膜新生血管膜手术切除后的年龄相关性黄斑变性的连续治疗。本研究的目的是建立一个模型，以评估视网膜色素上皮细胞移植到人布鲁赫膜在体外。我们研究了培养的胎儿人视网膜色素上皮细胞定植于人尸体布鲁赫膜的能力，确定了形成单层细胞和显示顶端微绒毛和紧密连接所需的孵育时间，并评估了基底膜的产生。通过睫状体平坦部切开新鲜摘除的（小于48小时）人眼，并取出前段、玻璃体和视网膜。用手术海绵清除天然视网膜色素上皮。用直径为7.0 mm的环钻对黄斑处的Bruch膜和脉络膜进行环钻，然后与1/2 ml Dulbecco改良Eagle培养基+15%胎牛血清+碱性成纤维细胞生长因子（1 ng/ml）和浓度为242，000个细胞/ml的胎儿人视网膜色素上皮细胞孵育。将样本孵育1、4、6、8、12或24小时。将标本固定在一半强度的Karnovsky固定剂中，处理，并用扫描和透射电子显微镜进行分析。视网膜色素上皮在不同孵育时间对黄斑清创标本有不同程度的覆盖。1小时后，细胞开始附着并变平（中位数百分比覆盖率：78%）。胎儿视网膜色素上皮细胞对布鲁赫膜的覆盖程度在不同的标本之间差异很大。4-6小时后，细胞以单层覆盖整个清创表面（中位覆盖百分比：4小时为97.2%，6小时为99.8%）。细胞间紧密连接，顶端微绒毛少。细胞侧缘相对于布鲁赫膜呈倾斜方向，细胞核伸长，核仁突出，与布鲁赫膜平行。6-8小时后，细胞开始变成六边形（8小时时的中值覆盖百分比：99.97%）。附着于内胶原层的细胞比附着于残留的天然基底膜的细胞更平坦。在12和24小时，观察到六边形、紧密连接和顶端微绒毛的表达更频繁（中位覆盖百分比：12小时为99.87%，24小时为100%）。在这些时间点未观察到新形成的基底膜。在比较存在和不存在天然RPE基底膜的情况下的附着的单独实验中，天然视网膜色素上皮基底膜的存在促进了细胞的早期附着和正常形态的更快速表达。这种体外系统提供了一种可重复的方法来研究视网膜色素上皮细胞对正常和患病的人布鲁赫膜的粘附。(C)出版社：Academic Press Limited。
Retinal pigment epithelium transplantation has been proposed as adjunctive treatment for age-related macular degeneration following surgical excision of choroidal neovascular membranes. The goal of this study was to develop a model to evaluate retinal pigment epithelium transplantation onto human Bruch's membrane in vitro. We investigated the ability of cultured fetal human retinal pigment epithelium to colonize human cadaver Bruch's membrane, determined the incubation time needed to form a monolayer and to exhibit apical microvilli and tight junctions, and assessed the production of basement membrane. Freshly enucleated (less than 48 hours old) human eyes were cut through the pars plana, and the anterior segment, vitreous, and retina were removed. The native retinal pigment epithelium was debrided with a surgical sponge. Bruch's membrane and choroid at the macula were trephined with a 7.0 mm diameter trephine and then incubated with 1/2 mi of Dulbecco's modified Eagle's medium +15% fetal calf serum + basic fibroblast growth factor (1 ng ml(-1)), and fetal human retinal pigment epithelium at a concentration of 242,000 cells ml(-1). Specimens were incubated for 1, 4, 6, 8, 12, or 24 hours. The specimens were fixed in half strength Karnovsky's fixative, processed, and analysed with scanning and transmission electron microscopy. The retinal pigment epithelium covered the debrided macular specimens to different degrees at different incubation times. After 1 hour, the cells started to attach and flatten (median percent coverage: 78%). The extent of Bruch's membrane coverage by fetal retinal pigment epithelium varied greatly between specimens. After 4-6 hours, the cells covered the entire debrided surface in a monolayer (median percent coverage: 97.2% at 4 hours, 99.8% at 6 hours). Tight junctions were observed, and the cells had few apical microvilli. The lateral cell borders were obliquely oriented with respect to Bruch's membrane, and the nuclei were elongated, exhibited prominent nucleoli, and were oriented parallel to Bruch's membrane. After 6-8 hours, cells started to become hexagonal (median percent coverage at 8 hours: 99.97%). Cells attached to the inner collagenous layer tended to be flatter than cells attached to residual native basement membrane. At 12 and 24 hours, expression of hexagonal shape, tight junctions, and apical microvilli were observed more frequently (median percent coverage: 99.87% at 12 and 100% at 24 hours). No newly formed basement membrane was observed at these time points. In separate experiments comparing attachment in the presence and absence of native RPE basement membrane, the presence of native retinal pigment epithelial basement membrane promoted the early attachment of the cells and more rapid expression of normal morphology. This in vitro system provides a reproducible way to study the adherence of retinal pigment epithelium to normal and diseased human Bruch's membrane. (C) 1998 Academic Press Limited.