The Biological Characterization of Cultured Rabbit Corneal Endothelial Cells

1. Yiqian Sun
2. Jing Hong

Department of Ophthalmology,Third Hospital of Peking University,Beijing,China

Purpose To establish the cultivating and identifying approach of the rabbit corneal endothelial cells (RCECs) and detect the biological characteristics after passages, lay foundation for corneal endothelial tissue engineering.

Method RCECs were isolated from Descemet's membrane which has been peeled off completely and digested with trypsin.The growth state and morphology of RCECs was observed by inverted phase-contrast microscope and alizarin red staining.RCECs were identified from cellular morphology,gene and protein level: collagen type IV α2(COL4A2)、vascular endothelial growth factor receptor 2 (FLK1) 、Na⁺-K⁺ATPase alpha 1 subunit (ATP1A1)、aquaporin 1 (AQP1)、 voltage-dependent anion channels（VDACs）were detected by Reverse Transcription-Polymerase Chain Reaction(RT-PCR).The expression and distribution of neurone specific enolase(NSE) Na⁺-K⁺ATPase、zonula occludens-1(Zo-1) were detected by immunocytochemistry under fluorescence microscope.The grawth curve was drawn by MTT assay to compare the proliferation activity after passages. Na⁺-K⁺ATPase activity of different generations cells was measured by ATPase kit.

Result Most of the cultured cells adhered in 24h, fused in 2-3 days with hexagon, but the shape changes after passages.Alizarin red staining showed a clear cellullar morphology which was similar to the cells on fresh corneal tissue .Target genes of COL4A2、ATP1A1 etc.were expressed firmly.NSE, Na⁺-K⁺ATPase,Zo-1 positive cells were respectively observed under laser confocal scanning microscopy.MTT result showed the decrease of cell proliferation activity. Quantitative results also showed the decrease of Na+-K+ATPase activity after passage.

Conclusion The approach of the culturing and idtifying RCECs was successful. The proliferation activity and Na⁺-K⁺ATPase activity was decreasing after passages.