Ohji M, SundarRaj N, Hassell J R, Thoft R A
Department of Ophthalmology, University of Pittsburgh, PA 15213.
Invest Ophthalmol Vis Sci. 1994 Feb;35(2):479-85.
Collagen gels may prove to be potential carriers for transplantation of cultured corneal epithelial cells. The purpose of this study was to evaluate the suitability of collagen gels in comparison with corneal stromal blocks as the substrate to support the growth of human corneal epithelial cells in culture and the synthesis and deposition of the basement membrane components by these cells.
Corneal epithelial sheets, freed from the culture dishes using Dispase II (Boehringer Mannheim, Indianapolis, IN), were cultured on corneal stromal blocks. Deposition of laminin, type IV collagen, type VII collagen, and perlecan (heparan sulfate proteoglycan) were evaluated immunohistochemically after 4 days, 7 days, 2 weeks, and 3 weeks. Human limbal explant cultures were established on collagen gels prepared from bovine type I collagen with or without addition of cultured human corneal fibroblasts. After 1, 2, 3, and 4 weeks, the deposition of the basement membrane components was evaluated immunohistochemically.
Corneal epithelial cells, cultured on corneal stromal blocks as well as on collagen gels with or without fibroblasts, deposited laminin, type IV collagen, perlecan, and type VII collagen at the interface of the cells and the substrates. However, different substrates differentially influenced the temporal pattern of the deposition of various basement membrane components. On the stromal blocks, deposition of laminin, type IV collagen, and perlecan by the epithelial cells was evident at 1 week. Type VII collagen was detected at 2 weeks. On the collagen gels with fibroblasts, deposition of laminin, type IV collagen and perlecan was detectable at 1 week. In the epithelial cultures on the collagen gels without fibroblasts, only perlecan was detectable at 1 week. At 2 weeks, all of the basement membrane components, including type VII collagen were detectable on the collagen gels, either with or without fibroblasts.
Human corneal epithelium cultured on collagen gels or on corneal stromal blocks can synthesize and deposit basement membrane components, including laminin, type IV collagen, type VII collagen, and perlecan within 2 weeks in culture. Therefore, collagen gels may serve as potential carriers for human corneal epithelial transplantation.
胶原凝胶可能被证明是培养的角膜上皮细胞移植的潜在载体。本研究的目的是评估胶原凝胶与角膜基质块相比作为支持人角膜上皮细胞在培养中生长以及这些细胞合成和沉积基底膜成分的基质的适用性。
使用Dispase II（勃林格殷格翰公司，印第安纳波利斯，印第安纳州）从培养皿中分离出的角膜上皮片，在角膜基质块上培养。在4天、7天、2周和3周后，通过免疫组织化学评估层粘连蛋白、IV型胶原、VII型胶原和基底膜聚糖（硫酸乙酰肝素蛋白聚糖）的沉积情况。在添加或不添加培养的人角膜成纤维细胞的情况下，用牛I型胶原制备胶原凝胶，建立人角膜缘外植体培养物。在1、2、3和4周后，通过免疫组织化学评估基底膜成分的沉积情况。
在角膜基质块以及添加或不添加成纤维细胞的胶原凝胶上培养的角膜上皮细胞，在细胞与基质的界面处沉积了层粘连蛋白、IV型胶原、基底膜聚糖和VII型胶原。然而，不同的基质对各种基底膜成分沉积的时间模式有不同的影响。在基质块上，上皮细胞在1周时明显沉积层粘连蛋白、IV型胶原和基底膜聚糖。在2周时检测到VII型胶原。在添加成纤维细胞的胶原凝胶上，在1周时可检测到层粘连蛋白、IV型胶原和基底膜聚糖的沉积。在不添加成纤维细胞的胶原凝胶上的上皮培养物中，在1周时仅可检测到基底膜聚糖。在2周时，无论有无成纤维细胞，在胶原凝胶上均可检测到所有基底膜成分，包括VII型胶原。
在胶原凝胶或角膜基质块上培养的人角膜上皮在培养2周内可合成并沉积基底膜成分，包括层粘连蛋白、IV型胶原、VII型胶原和基底膜聚糖。因此，胶原凝胶可作为人角膜上皮移植的潜在载体。
