Sawhney R S
Department of Medicine and Biochemistry, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612.
Invest Ophthalmol Vis Sci. 1993 Jun;34(7):2195-202.
To identify the types of procollagen that are synthesized by cultured bovine lens epithelial cells and to determine what types of post-translational modifications are involved in procollagen biosynthesis and what patterns of procollagen gene expression occur in these cells.
The epithelial nature of the cultured cells was confirmed with transmission electron and phase contrast microscopy. To label collagen, primary or secondary monolayer cultures were pulsed with [3H] proline for various periods of time. Procollagens were identified by immunoprecipitation with specific antibodies, pepsin digestion, and electrophoretic separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Procollagen messenger RNA was identified by Northern blot analysis using specific DNA probes.
In the normal lens epithelium, cells are attached to each other by tight junctions punctuated with desmosomes. After the proteins were labeled, they were immunoprecipitated with antibodies specific to collagen precursors and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to further confirm their identification as types I and III procollagen or processed forms of these proteins. The secretion of procollagen was inhibited by treatment with 2,2'-dipyridyl and bands of underhydroxylated procollagen were observed in the cell layer after electrophoresis. The sizes of messenger RNA observed were 6.9 kb and 5.4 kb for type alpha 1(I), 5.0 kb for type alpha 2(I), and 5.3 kb for the type III transcripts.
This is the first report that lens epithelial cells express fibrillar procollagen genes, making the lens capsule a unique ocular basement membrane.
鉴定培养的牛晶状体上皮细胞合成的前胶原类型，确定前胶原生物合成中涉及哪些类型的翻译后修饰，以及这些细胞中前胶原基因表达的模式。
用透射电子显微镜和相差显微镜确认培养细胞的上皮性质。为标记胶原蛋白，将原代或传代单层培养物用[3H]脯氨酸脉冲处理不同时间。通过用特异性抗体进行免疫沉淀、胃蛋白酶消化以及在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上进行电泳分离来鉴定前胶原。使用特异性DNA探针通过Northern印迹分析鉴定前胶原信使RNA。
在正常晶状体上皮中，细胞通过有桥粒点缀的紧密连接相互附着。蛋白质标记后，用针对胶原前体的特异性抗体进行免疫沉淀，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离，以进一步确认它们被鉴定为I型和III型前胶原或这些蛋白质的加工形式。用2,2'-联吡啶处理可抑制前胶原的分泌，电泳后在细胞层中观察到羟化不足的前胶原条带。观察到的信使RNA大小，α1(I)型为6.9 kb，α2(I)型为5.0 kb，III型转录本为5.3 kb。
这是首次报道晶状体上皮细胞表达纤维状前胶原基因，使晶状体囊成为独特的眼基底膜。
