Wei Z G, Lin T, Sun T T, Lavker R M
Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Invest Ophthalmol Vis Sci. 1997 Mar;38(3):753-61.
This study investigated the in vivo differentiation of conjunctival keratinocytes.
Keratinocytes from the fornical region of the conjunctival epithelium were isolated and plated at low density (5 x 102 per 100-mm dish) in Dulbecco's minimum essential medium containing 20% fetal bovine serum in the presence of mitomycin C-treated 3T3 feeder cells. At this density, only single, isolated cells were attached after overnight culture. Eight days later, small, well-isolated colonies separated from one another by the feeder cells were detached as a sheet from the dish and were injected subcutaneously into the flanks of BALB/c athymic mice through an 18-gauge needle. Within a day, a small firm nodule appeared at the site of injection. At different time points, the animals were killed, and the nodules were excised for morphologic, histogeometric, and cell kinetic analyses.
Each implanted colony derived from a single cell gave rise to a single epithelial cyst lined with a reconstituted stratified epithelium. Goblet-like cells loaded with periodic acid-Schiff-positive cytoplasmic granules began to appear singularly in some of the cysts by day 8 postimplantation and were observed in approximately 85% of the cysts by day 14.
Because the cysts formed were derived from clonal populations of epithelial cells and the majority of cysts had a mixed keratinocyte-goblet cell phenotype, these results suggest strongly the existence of a bipotent precursor cell in conjunctival epithelium that can give rise to both goblet and nongoblet cells. This system can be used to study factors that can influence the commitment of pluripotent epithelial stem cells to divergent pathways of differentiation.
本研究调查结膜角质形成细胞的体内分化情况。
从结膜上皮穹窿部分离角质形成细胞，以低密度（每100毫米培养皿5×10²个细胞）接种于含20%胎牛血清的杜尔贝科改良伊格尔培养基中，同时存在丝裂霉素C处理过的3T3饲养细胞。在此密度下，过夜培养后只有单个分离的细胞附着。8天后，被饲养细胞彼此分隔开的小的、分离良好的集落作为一片从培养皿上分离下来，通过18号针头皮下注射到BALB/c无胸腺小鼠的侧腹。一天内，注射部位出现一个小的坚实结节。在不同时间点处死动物，切除结节进行形态学、组织几何学和细胞动力学分析。
每个源自单个细胞的植入集落形成一个单一的上皮囊肿，内衬重构的复层上皮。植入后第8天，一些囊肿中开始单个出现充满过碘酸希夫阳性细胞质颗粒的杯状样细胞，到第14天，约85%的囊肿中可观察到这种细胞。
由于形成的囊肿源自上皮细胞的克隆群体，且大多数囊肿具有角质形成细胞-杯状细胞混合表型，这些结果强烈提示结膜上皮中存在一种双能前体细胞，它可产生杯状细胞和非杯状细胞。该系统可用于研究影响多能上皮干细胞向不同分化途径定向分化的因素。
