Tezel Tongalp H, Del Priore Lucian V, Kaplan Henry J
Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA.
Invest Ophthalmol Vis Sci. 2004 Sep;45(9):3337-48. doi: 10.1167/iovs.04-0193.
An earlier study showed that age-related changes in the inner collagen layer (ICL) inhibit RPE cell repopulation of human Bruch's membrane. The present study was undertaken to determine the effect of cleaning and/or an extracellular matrix (ECM) protein coating on the reattachment, apoptosis, proliferation, and final surface coverage of the transplanted RPE cells.
Explants of aged Bruch's membrane with ICL exposed were prepared from five human cadaveric eyes (donor ages, 69-84 years) and treated with Triton X-100 and/or coated with a mixture of laminin (330 microg/mL), fibronectin (250 microg/mL), and vitronectin (33 microg/mL). Viable human fetal and ARPE-19 cells (n = 15,000) were plated onto the surface and the RPE reattachment, apoptosis, and proliferation ratios were determined on the modified surfaces. Cells were cultured up to 17 days to determine the surface coverage. Ultrastructure of the modified Bruch's membrane and RPE morphology were studied with transmission and scanning electron microscopy.
Reattachment ratios of fetal human RPE and ARPE-19 cells were similar on aged ICL (41.5% +/- 1.7% and 42.9% +/- 2.7%, P > 0.05). The reattachment ratio increased with ECM protein coating and decreased with detergent treatment. Combined cleaning and coating restored the reattachment ratio of the fetal RPE cells, but failed to increase the reattachment ratio of ARPE-19 cells. The highest apoptosis was observed on untreated ICL. Cleaning and the combined procedure of cleaning and ECM protein coating decreased fetal RPE cell apoptosis. Only RPE cells plated on cleaned or cleaned and ECM-coated ICL demonstrated proliferation that led to substantial surface coverage at day 17.
Age-related changes that impair RPE repopulation of Bruch's membrane can be significantly reversed by combined cleaning and ECM protein coating of the ICL. Development of biologically tolerant techniques for modifying the ICL in vivo may enhance reattachment of the RPE and its repopulation of aged ICL.
一项早期研究表明，内胶原层（ICL）中与年龄相关的变化会抑制人 Bruch 膜上视网膜色素上皮（RPE）细胞的重新填充。本研究旨在确定清洗和/或细胞外基质（ECM）蛋白包被对移植的 RPE 细胞重新附着、凋亡、增殖及最终表面覆盖情况的影响。
从五例人尸体眼（供体年龄 69 - 84 岁）制备暴露有 ICL 的老年 Bruch 膜外植体，用 Triton X - 100 处理和/或用层粘连蛋白（330 μg/mL）、纤连蛋白（250 μg/mL）和玻连蛋白（33 μg/mL）的混合物包被。将存活的人胎儿和 ARPE - 19 细胞（n = 15,000）接种到表面，测定在改良表面上 RPE 细胞的重新附着、凋亡和增殖率。细胞培养长达 17 天以确定表面覆盖情况。用透射和扫描电子显微镜研究改良的 Bruch 膜的超微结构和 RPE 形态。
人胎儿 RPE 细胞和 ARPE - 19 细胞在老年 ICL 上的重新附着率相似（分别为 41.5%±1.7%和 42.9%±2.7%，P > 0.05）。重新附着率随 ECM 蛋白包被而增加，随去污剂处理而降低。联合清洗和包被可恢复胎儿 RPE 细胞的重新附着率，但未能提高 ARPE - 19 细胞的重新附着率。在未处理的 ICL 上观察到最高的凋亡率。清洗以及清洗和 ECM 蛋白包被的联合操作可降低胎儿 RPE 细胞凋亡。只有接种在清洗过的或清洗并经 ECM 包被的 ICL 上的 RPE 细胞表现出增殖，在第 17 天导致大量的表面覆盖。
通过联合清洗和 ICL 的 ECM 蛋白包被，可显著逆转损害 Bruch 膜上 RPE 重新填充的与年龄相关的变化。开发用于体内修饰 ICL 的生物耐受性技术可能会增强 RPE 的重新附着及其在老年 ICL 上的重新填充。
