Biological characterization of cultured rabbit corneal endothelial cells / 中华实验眼科杂志

ABSTRACT

Background How to harvest the purified corneal endothelial seed cells is very important for the corneal tissue engineering technology. Herein,to establish a good culture method and effective identification method of corneal endothelial cells ( CECs) is the key. Objective Present study wag to establish the cultivating and identifying approach of the rabbit CECs and detect the biological characteristics of passaged cells. Methods Rabbits CECs were isolated from Descemet's membrane peeled off completely in 30 New Zealand white rabbits and then digested with 0. 25% trypsin-0. 02% EDTA and primarily cultured in CECs medium containing 15% fetal bovine serum. The growth situate and cellular morphology of rabbit CECs were observed under the inverted phase-contrast microscope following the alizarin red staining. Rabbit CECs were identified by cellular morphology as well as in gene and protein level, including the detection of expressions of collagen type IV α2(COL4A2) ,vascular endothelial growth factor receptor 2 ( FLK1 ) , Na+-K+ ATPase alpha 1subunit ( ATP1 A1 ) , aquaporin 1( AQP1 ) , voltage-dependent anion channels ( VDACs) 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 also detected by immunocytochemistry under the fluorescence microscope. The proliferation activity of the passage cells was dynamically observed by MTT assay,and Na+-K+ ATPase activity of different generations cells was detected by ATPase kit. Results Majority of the primarily cultured cells adhered in 24 hours, infused in 2-3 days with the hexagon shape in appearance. The morphology of the cells was very varied with passage. Alizarin red staining showed a well- defined and well-lined cellular morphology similar to the corneal cells in vivo. Target genes of C0L4A2, FLK1, ATPIAI ,AQPI and VDACs were positively expressed in the cells. However,the expression of CK12 was absent in the cells. NSE, Na+-K+ ATPase, ZO-1 positive cells were respectively observed under the laser confocal scanning microscopy. MTT results showed a gradually low growth curve with the passage. Quantitative results also revealed that the Na+-K+ ATPase activity was gradually declined in different generations of cells ( F = 77. 174, P = 0. 000 ). Conclusion The enzyme digestion is a better approach of isolating and culturing comeal endothelial cells. Cultured cells can be identified by cells staining, gene expression and protein level. Earlier generation of CECs are ideal seed cells for cornea tissue engineering.