Effects Of Aging On The Biological Characteristics Of Cultured Limbal Stem Cells On The Corneal Stroma As A Carrier In Vitro

Objectives By culturaling different ages rabbits LSCs in different age rabbits corneal stroma carrier to observe the change of aging on the biological characteristics of LSCs seeded in allogeneic corneal stroma, to investigate the effects of aging on proliferation capacity of limbal stem cells seeded in corneal stroma carrier; by detecting the effects of aging of expression ofΔNp63 and ABCG2 protein of LSCs seeded in corneal stroma to investigate the relevancy with aging of stem cell cultured in carrier, and to develop new way for corneal seeds cell selection of tissue engineering.Methods Experimental study on the control. Limbal epithelium harvested from New Zealand rabbits of different ages were enzymatically cleft into single cell suspension and cultured, and limbal stem cells were seeded at a same cell density in the corneal stroma of different age of New Zealand white rabbits. The growth status of LSCs seeded in the corneal stroma was observed while identification was performed using in situ hybridization, image was analyzed in IPP5.1 software, cell cycle was examined by flow cytometry, the expression ofΔNp63 and ABCG2 was assessed by RT-PCR technique and the experimental data was analysed by ANOVA of factorial design.Results The young LSCs seeded in different age corneal stroma proliferates faster than other group, the delitescence is short, smaller shape, round, ellipse, polygon, and form a larger colony. The serial subcultivation LSCs in corneal stroma carrier is identificated by ABCG2 and P63 in situ hybridization, most cells present positive reaction, the endochylema presents buffy and the nucelus presents blue. The difference in the level of LSCs donor age has statistical significance, as the ratio of proliferating cells and and the expression ofΔNp63 and ABCG2 protein was the highest in the young rabbits compared with the middle-aged and elder rabbits; the difference in the level of corneal stroma donor age and the interation of LSCs and corneal stroma has no statistical significance.Conclusions The proliferation capacity of limbal stem cells seeded in allogeneic corneal stroma was shown to decrease with aging of the donor of limbal stem cells. The expression ofΔNp63 and ABCG2 protein decreases with aging of the donor of limbal stem cells, which suggests thatΔNp63 and ABCG2 protein may play an important role in the aging process of limbal stem cell cultured by cornea stroma.