Phenotypic heterogeneity of retinal pigment epithelial cells in vitro and in situ
Phenotypic heterogeneity of retinal pigment epithelial cells in vitro and in situ
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DOI：
10.1006/exer.1996.0008
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发表时间：
1996-01-01
影响因子：
3.4
通讯作者：
McKay, BS
中科院分区：
文献类型：
--
作者：
Burke, JM;Skumatz, CMB;McKay, BS
我们之前已经开发了基于不同细胞黏附的方法，从人RPE培养物中选择两种不同表型的细胞，上皮样细胞和梭形细胞。在这里，我们考虑细胞形状的差异是否与蛋白质酪氨酸磷酸化的差异相关，因为众所周知，磷酸化的升高扰乱了附着连接的稳定性，附着连接是上皮表型的主要决定因素。在两种表型的培养物中，我们发现融合后培养物的酪氨酸磷酸化水平较低，用磷酸酶抑制剂处理后，酪氨酸磷酸化蛋白的补体相同。然而，与上皮样细胞不同，梭形RPE不能将磷蛋白定位到细胞外围的连接部位。我们还对原代和传代培养的RPE进行了重新评估，以寻找更多的细胞形状变体。在相同的培养物中鉴定出几种不同的表型。它们需要几个星期的融合才能在原代和传代培养中发育，但在发育后几个月保持稳定。由于移植的RPE细胞在相同的培养环境中表现出几种形状变体，我们在原位观察了牛RPE细胞，以确定这些细胞是否在一些可能影响细胞形状的细胞表面和细胞骨架蛋白方面是异质性的。环状肌动蛋白微丝束和闭合连接蛋白ZO-1在单层细胞中的分布比较均匀，但中间丝蛋白波形蛋白和磷酸酪氨酸在细胞周围的表达在不同细胞之间存在差异。因此，尽管RPE单层外观大体均匀，但原位RPE单层可被认为是结构不同的细胞的镶嵌，在体外培养时可产生表型不同的亚群。(C)1996年学术出版社有限公司
We have previously developed methods based upon differential cell adhesion to select for cells of two different phenotypes, epithelioid and fusiform, from cultures of human RPE. Here we considered whether the differences in cell shape correlated with differences in protein tyrosine phosphorylation since it is known that elevated phosphorylation perturbs the stability of the adherens junction, a major determinant of epithelial phenotype. In cultures of both phenotypes we found low tyrosine phosphorylation levels in postconfluent cultures, and the same complement of tyrosine phosphoproteins after treatment with a phosphatase inhibitor. However, in contrast to epithelioid cells, fusiform RPE failed to localize the phosphoproteins to junctional sites on the cell periphery. We also re-evaluated primary and passaged RPE cultures for additional cell shape variants. Several discrete phenotypes were identified within the same cultures. They required several weeks at confluency to develop in primary as well as in passaged cultures, but after developing they remained stable for months. Since explanted RPE cells manifest several shape variants in an identical culture environment we examined bovine RPE cells in situ to determine whether the cells were heterogeneous with regards to some cell surface and cytoskeletal proteins that might contribute to cell shape. Circumferential actin microfilament bundles and the occluding junction protein ZO-1 had fairly uniform distributions among cells in the monolayer, but the intermediate filament protein vimentin and the pericellular expression of phosphotyrosine varied among individual cells. Therefore, despite its grossly homogeneous appearance, the RPE monolayer in situ might be considered a mosaic of structurally heterogeneous cells which can give rise to phenotypically-distinct subpopulations when propagated in vitro. (C) 1996 Academic Press Limited