Characterization Of Human Corneal Epithelial Cell Line And Reconstruction Of Corneal Epithelium In Vitro

Cornea is composed of epithelium, Bowman,stroma, descemet and endothelium and play irreplaceable role to prevent water loss and pathogen invasion. Since the epithelium of cornea contacts with external environment directly,it is prone to be hurted and infected resulted in structure and function abnormity which perhaps bring on ireversible lesions or even cause bind corneal epithelium.There are many paients caused by bind corneal epithelium in four million people in China and 55 million bind corneal patients. At present, corneal epithelial transplantation is the sole method to cure bind corneal epithelium, however most of the patients could not accecpt corneal epithelial transplantation for the serious shortage of donated corneas. Corneal epithelial tissue engineering, as succedaneum of corneal epithelial, is a new feasible method to solve the lack of corneal material and also the hope of bind corneal epithelium patients. In order to bulid corneal epithelial tissue engineering vitro rebuild conditions, current study plan to identify human corneal epithelial clones that has been built and study human corneal epithelial tissue vitro rebuild utilizing air-liquid interface culture method.First of all,current thesis coducted preliminary identification on established human corneal epithelial clone using microscope observation, growth characteristics and chromosome analysis, BALB/c tumorigenicity test. According to the results of optical microscope observation, vitro-cultured human corneal epithelial own higher transparency and plump conformation as stable epithelial-like cell morphology that could passage when cultured 3 days.Proliferation time of the cells is 45.42 h indicated keep strong ability to cleavage. Chromosome analysis showed that the cell lines have their Characteristic chromosome number in 46, although some cells are chromosomal aneuploidy.Karyotype analysis showed that the cells have a typical diploid karyotype(12m+22s+10t+XX).Cells subcutaneous injection of the cell line has not caused tumorigenesis implied no any tumorigenicity as tumorigenicity test results showed. Thus it can be seen current cells of human corneae epithelium cell line could be used as seed cells of vitro-rebuilt human corneal epithelial tissue engineering.Meanwhile, current article conducted preparing research eliminating amniotic epithelium.To ingest fresh amnion using 0.25% Trypsin-0.02% EDTA mixture, washing utilizing D-Hanks solution after cleaning pellicle to gain amniotic eptthelium to served as carrier bracket of vitro-rebuilt human corneal epithelial tissue engineering.At last, vitro-rebuilt human corneal epithelial tissue engineering is studied using air-liquid interface culture method, non-transfected, non-oncogenic cells of human corneal epithelial cell line as seed cells and cleaning pellicle amnion as carrier bracket. According to the results of optical microscope, corneal epithelial cells attached to the leather of amniotic membrane could form single layer for 24 h and could form 5-8 layers at the 9th day. HE staining results of human corneal epithelial tissue engineering slices showed it could form stratified epithelium consisted of 3 layers corneal epithelial cells after 5 days, stratified epithelium consisted of 4-5 layers corneal epithelial cells after 7 days, and stratified epithelium with preferable transparency and connectivity, consisted of 5-8 layers corneal epithelial cells after 9 days. According to the immunofluorescence test results, the seed cells possess Keratin K3 positive expression which is the marker proteins of corneal epithelial cell specificity, meanwhile expresses integrinβ1.Based on scanning electron microscopy results, the seed cells still keep original form of corneal epithelial cells, possess microvilli structure at surface and forms continuous dense layer of corneal epithelial cells.These results indicate that seed cells still could maintain the properties of corneal epithelial cells after vitro reconstrction,form 5-8 layers of stratified corneal epithelium similar to corneal epithelial cells and possess anchor connective ability. In conclusion, current article successfully rebuilt tissue engineering human corneal epithelial similar to forms and proporties of vivo human corneal epithelial using air-liquid interface culture method, non-transfected, non-oncogenic cells of human corneal epithelial cell line as seed cells and cleaning pellicle amnion as carrier bracket and supply reference for large scale vitro-reconstruction, clinical application and vitro-reconstruction of tissue engineering of human whole cornea.