Department of Ophthalmology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima City, Hiroshima, Japan.
Invest Ophthalmol Vis Sci. 2014 Jan 7;55(1):125-33. doi: 10.1167/iovs.13-12642.
To examine the effects of neural cells on the stratification of and junctional protein expression by corneal epithelial cells with a coculture system.
PC12 cells induced to undergo neuronal differentiation or rat trigeminal nerve cells were cultured together with simian virus 40-transformed human corneal epithelial (HCE) cells on opposite sides of a collagen vitrigel membrane. Stratification of HCE cells was examined by immunofluorescence analysis with antibodies to zonula occludens-1. Expression of junctional proteins in HCE cells was assessed by RT-PCR and immunoblot analyses.
The presence of neural cells (PC12 cells or trigeminal neurons) markedly promoted the stratification of HCE cells as well as increased the amounts of N-cadherin mRNA and protein in these cells. These effects of the neural cells were mimicked by conditioned medium prepared from differentiating PC12 cells or by the neuropeptides substance P and calcitonin gene-related peptide (CGRP). Furthermore, the stimulatory effects of trigeminal neurons on the stratification of and N-cadherin expression by HCE cells were inhibited by antagonists of substance P or of CGRP.
These results suggest that trigeminal neurons play an important role in the differentiation of corneal epithelial cells. Neuropeptides released from these neurons may thus regulate adhesion between corneal epithelial cells and thereby contribute to the establishment and maintenance of corneal structure and function.
利用共培养系统研究神经细胞对角膜上皮细胞分层及连接蛋白表达的影响。
将诱导分化的 PC12 细胞或大鼠三叉神经细胞与猴病毒 40 转化的人角膜上皮（HCE）细胞培养在胶原 vitrigel 膜的两侧。用针对封闭蛋白-1 的抗体通过免疫荧光分析来检测 HCE 细胞的分层。通过 RT-PCR 和免疫印迹分析评估 HCE 细胞中连接蛋白的表达。
神经细胞（PC12 细胞或三叉神经细胞）的存在显著促进了 HCE 细胞的分层，并增加了这些细胞中 N-钙黏蛋白 mRNA 和蛋白的含量。来自分化的 PC12 细胞的条件培养基或神经肽 P 物质和降钙素基因相关肽（CGRP）模拟了神经细胞的这些作用。此外，三叉神经神经元对 HCE 细胞分层和 N-钙黏蛋白表达的刺激作用被 P 物质或 CGRP 的拮抗剂所抑制。
这些结果表明三叉神经神经元在角膜上皮细胞的分化中起重要作用。这些神经元释放的神经肽可能通过调节角膜上皮细胞之间的黏附，从而有助于角膜结构和功能的建立和维持。
