Crawford B J, Crawford T J
Tissue Cell. 1984;16(6):885-908. doi: 10.1016/0040-8166(84)90069-7.
In clonal culture, colonies of 3-4 week old chick retinal pigmented epithelial cells exhibit Alcian Blue positive extracellular matrix (ECM) material on the surface of the cells. Alcian blue positive ECM is located between undifferentiated cells at the edges of the disc-shaped colonies and beneath the differentiated cells in the colony center. The latter material is associated with the basement membrane. The staining properties suggest that glycosaminoglycans (GAG) are present in these regions. Extraction of GAG from homogenates of colonies, followed by electrophoresis on cellulose acetate strips, results in three bands with mobilities similar to those of hyaluronic acid, heparan sulfate, and chondroitin sulfate, respectively. All three bands label with [3H]glucosamine, and the last two also label with [35S]sulfate. The composition appeared to differ when colonies were grown in different media. Digestion of the GAG preparations with various enzymes suggests that bands II and III represent heparan sulfate and chondroitin sulfate, respectively, in colonies grown in Ham's F10g medium. The composition of band I is as yet undetermined. In minimal Eagle's medium (MEM), bands I and III consisted of hyaluronic acid and chondroitin sulfate, respectively, while band II had properties suggestive of a copolymer of heparan sulfate and an unidentified GAG. Cells release only one [3H]glucosamine-labelled GAG into the medium. This material has a mobility similar to hyaluronic acid and is digested by Streptomyces hyaluronidase, suggesting that it is hyaluronic acid. Staining with Alcian Blue at different pH suggests that it may represent the material associated with the upper surface of the cells. Some of the ECM located between the undifferentiated cells and associated with the basement membrane in the differentiated regions of the colonies stains with Alcian Blue at pH 1.0 and 0.2 suggesting that it may contain GAGs found in bands I and II. Colonies treated with medium containing 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of GAG synthesis, for 48 hr showed a reduced Alcian Blue staining of the ECM in the undifferentiated regions. After 72 hr of treatment with DON, the undifferentiated cells had detached from the plate, whereas the differentiated cells remained intact. The results suggest that the GAG may be involved in cellular adhesion, particularly of the undifferentiated cells.
在克隆培养中，3 - 4周龄鸡视网膜色素上皮细胞的集落，在细胞表面呈现出阿尔新蓝阳性的细胞外基质（ECM）物质。阿尔新蓝阳性的ECM位于盘状集落边缘的未分化细胞之间以及集落中心的分化细胞之下。后者的物质与基底膜相关。染色特性表明这些区域存在糖胺聚糖（GAG）。从集落匀浆中提取GAG，然后在醋酸纤维素条上进行电泳，结果得到三条带，其迁移率分别与透明质酸、硫酸乙酰肝素和硫酸软骨素的迁移率相似。所有三条带都用[³H]葡萄糖胺标记，后两条带也用[³⁵S]硫酸盐标记。当集落在不同培养基中生长时，其组成似乎有所不同。用各种酶消化GAG制剂表明，在哈姆F10g培养基中生长的集落中，带II和带III分别代表硫酸乙酰肝素和硫酸软骨素。带I的组成尚未确定。在最低限度的伊格尔培养基（MEM）中，带I和带III分别由透明质酸和硫酸软骨素组成，而带II具有硫酸乙酰肝素和一种未鉴定的GAG共聚物的特性。细胞仅将一种[³H]葡萄糖胺标记的GAG释放到培养基中。这种物质的迁移率与透明质酸相似，并被链霉菌透明质酸酶消化，表明它是透明质酸。在不同pH值下用阿尔新蓝染色表明，它可能代表与细胞上表面相关的物质。集落中位于未分化细胞之间并与分化区域的基底膜相关的一些ECM，在pH 1.0和0.2时用阿尔新蓝染色，表明它可能含有带I和带II中的GAG。用含有6 - 重氮 - 5 - 氧代 - L - 正亮氨酸（DON）（一种GAG合成抑制剂）的培养基处理集落48小时，未分化区域的ECM阿尔新蓝染色减少。用DON处理72小时后，未分化细胞从平板上脱落，而分化细胞保持完整。结果表明，GAG可能参与细胞黏附，特别是未分化细胞的黏附。
