Zhu Ying-Ting, Hayashida Yasutaka, Kheirkhah Ahmad, He Hua, Chen Szu-Yu, Tseng Scheffer C G
Tissue Tech, Inc., Ocular Surface Center, and Ocular Surface Research and Education Foundation, Miami, Florida, USA.
Invest Ophthalmol Vis Sci. 2008 Sep;49(9):3879-86. doi: 10.1167/iovs.08-1693. Epub 2008 May 23.
Human corneal endothelial cell (HCEC) proliferation is controlled by HCEC junctions, but the mechanism of proliferation remains unknown. The authors sought to characterize adherent junction components of in vivo HCECs and to compare their gene expression and their proliferative potential with those of in vitro counterparts.
Stripped human Descemet membranes were digested with collagenase A, and the resultant HCEC aggregates were cultured for 7, 14, and 21 days in supplemented hormonal epithelial medium (SHEM). The growth of HCEC monolayers was monitored by BrdU labeling performed 24 hours before termination. In vivo and in vitro HCECs were subjected to immunostaining to FITC-phalloidin and antibodies to different junction components and BrdU. Their mRNA expressions were determined by RT-PCR.
In vivo HCECs expressed transcripts of N-, VE-, E-, and P-cadherins, alpha-, beta-, gamma-, and p120-catenins, and p190. In vitro HCEC counterparts also expressed all these mRNAs except P-cadherin. In vivo HCECs displayed continuous circular F-actin, N-cadherin, beta- and p120-catenins, and p190, discontinuous circular VE-cadherin bands at or close to cell junctions, and E-cadherin in the cytoplasm. Such an in vivo pattern was gradually achieved by in vitro HCECs at day 21 and was correlated with a progressive decline of BrdU labeling.
In vivo and in vitro HCECs displayed distinct protein cytolocalization of N-, VE-, and E-cadherins, beta- and p120-catenins, and p190. Progressive maturation of adherent junctions was associated with a decline of the proliferative potential. This information allows us to devise new strategies to engineer in vitro HCECs by targeting these components.
人角膜内皮细胞（HCEC）的增殖受HCEC连接调控，但其增殖机制尚不清楚。作者试图对体内HCEC的黏附连接成分进行表征，并将其基因表达及增殖潜能与体外培养的对应细胞进行比较。
用胶原酶A消化剥离的人Descemet膜，将所得的HCEC聚集体在添加激素的上皮细胞培养基（SHEM）中培养7、14和21天。在终止培养前24小时通过BrdU标记监测HCEC单层的生长情况。对体内和体外培养的HCEC进行免疫染色，分别使用异硫氰酸荧光素（FITC）标记的鬼笔环肽以及针对不同连接成分和BrdU的抗体。通过逆转录聚合酶链反应（RT-PCR）测定其mRNA表达。
体内HCEC表达N-钙黏蛋白、血管内皮（VE）-钙黏蛋白、E-钙黏蛋白、P-钙黏蛋白、α-连环蛋白、β-连环蛋白、γ-连环蛋白、p120-连环蛋白和p190的转录本。体外培养的HCEC对应细胞除P-钙黏蛋白外也表达所有这些mRNA。体内HCEC呈现连续的环形F-肌动蛋白、N-钙黏蛋白、β-连环蛋白、p120-连环蛋白和p190，在细胞连接处或其附近有不连续的环形VE-钙黏蛋白带，且E-钙黏蛋白位于细胞质中。体外培养的HCEC在第21天时逐渐形成这种体内模式，且这与BrdU标记的逐渐下降相关。
体内和体外培养的HCEC在N-钙黏蛋白、VE-钙黏蛋白、E-钙黏蛋白、β-连环蛋白、p120-连环蛋白和p190的蛋白细胞定位上存在差异。黏附连接的逐渐成熟与增殖潜能的下降相关。这些信息使我们能够通过针对这些成分设计新的策略来体外构建HCEC。
