An Experimental Study On Cloning Growth Of Rabbit Limbal Stem Cells With Rabbit Embryo Fibroblast Feeder Cells And Autograft Transplantation In Vitro

This research studies the growth and proliferation ,the biological identification of corneal limbal stem cells cocultivated with feeder cells-unproliferating rabbit embryo fibroblast cells in vitro,the efficacy of rabbit autograft corneal limbal stem cells cultivated on amniotic membrane transplanted to rabbit cornea which corneal limbal stem cells were badly damaged.provides experimental basis for limbal stem cells transplantation,which is a hopeful therapeutic method ocular surface diseases.There are two parts of this research.Part 1 An experimental study on cloning growth of rabbit limbal stem cells with rabbit embryo fibroblast feeder cellsObjective To examine the efficacy of mitomycin C on the proliferation of rabbit embryo fibroblast cells for different time and research the preparation of rabbit embryo fibroblast cells as feeder cells. To examine the growth and proliferation of rabbit limbal stem cells cocultivated with the rabbit embryo fibroblast cells treated by 10g/ml mitomycin C in vitro and study the efficacy of feeder cells. To identify and investigate propertie of clone of rabbit corneal limbal stem cells in presence of rabbit embryo fibroblast feeder cells in vitro. Methods The cells were cultured normally.After treated with 10g/ml mitomycin C for different time,the proliferation of rabbit embryo fibroblast cells were cultivated and examined by counting cells. Rabbit limbal stem cells were digested and cultivated with the rabbit embryo fibroblast cells treated by 10 g/ml mitomycin C in vitro .Then the cultivated cells with and without treated the rabbitembryo fibroblast cells examined by colony-forming efficiency (CFE) in primacy culture, first passage, second passage and third passage.Both of them were checked by CFE. Cultured rabbit corneal limbal stem cells were observed by light microscopy,scanning and transmission electron microscopy and indirect immunecytochemistry staining to identify them comprehensively. Results The rabbit embryo fibroblast cells could be passaged for long in this culture condition.they resembled by themselves in shape and could simply distinguish in appearance.The rabbit embryo fibroblast cells treated by lOug/ml mitomycin C for 2.5 hours still resembled in shape but lose proliferative capacity.There is no obvious change in number of cells during the culture, limbal stem cells with feeder cells grew as clones after seeded for 3~4 days and the number of cells in the clones are over four.After 5~6 days ,the number of cells in the clones increased greatly.Then the cells reached confluence after seeded for 10~12days.The average CFE: 11.51 + 1.27% in primary passage and 10.50+1.27% in first passage and 10.31 ?.03% in second passage and 9.33 + 0.96% in third passage of rabbit limbal stem cells with feeder cells; 10.16+ 0.68% in primary passage and 6.73 ?.69% in first passage and 2.67 + 0.95% in second passage and 0.90+0.19% in third passage of rabbit limbal stem cells without feeder cells.cloned cells were typical epithelium cells.There were a large quantity of micro villi, desmosomes and tonofilaments.Monoclonal antibodies AE1 and PCNA staining were positive in mostly cloned cells while AE5 was positive in minority of the cells. Conclusion The rabbit embryo fibroblast cells treated by lOug/ml mitomycin C for 2.5 hours are still living but lose proliferative capacity.They are suitable to be feeder cells for limbal stem cells in vitro culture. The stem cells cocultivated with feeder cells have higher proliferating capacity and the rabbit limbal stem cells treated by mitomycin C can help to keep the higher proliferating capacity of rabbit limbal stem cells, rabbit corneal limbal stem cells was cloned successfully by rabbit embryo fibroblast feeder cells.The cloned cells can maintain their characteristics of cornealepithelium and have high proliferative potency.Part 2 An experimental study on autograft transplantation with cultured limbal stem cells on amniotic membraneObjective To study the treatment of rabbit limbal stem cell deficiency by cultured limbal stem cells...