| Objectives By culturaling different ages rabbits LSCs in vitro to investigate the effect of aging on the biological characteristics of LSCs,to establish a vitro culture technique of rabbit LSCs;by testing the△Np63 expression of all ages rabbits LSCs to analyze the correlation between the expression of△Np63 protein of LSCs and aging,to see the physiological property of LSCs,to post the cause of visual function decline,to provide basis for clinical diagnosis and therapy.Methods New Zealand white rabbits with the age 3 months,1.5 years and 3 years were selected in this study and 6 rabbits for each group.The lamelliform corneal limbal tissue was isolated and cultured in DMEM/F12(1:1) medium containing 10%fetal bovine serum(FBS) using digestion cultural method.The second-generation LSCs were collected and identified by△Np63 immunofluorescence stain.RT-PCR technique and indirect immunofluorescence were performed to assess the change of expression of△Np63 protein with aging.Results Cultured LSCs grew well,proliferated exuberantly and presented the green fluorescence in the cell nucleus for△Np63.The proliferation capacity of LSCs and the number of LSCs were shown to decrease with aging.The older the age,the lower the△Np63 express. The fluorescence intensity of△Np63 protein expression was 121±22,94±23and 46±16 in 3-month-old,1.5-year-old and 3-year-old rabbits respectively,showing a statistically significant difference among these three groups(F=13.125,P＜0.05).The gray scale value of△Np63 mRNA expression was 0.7333±0.0117,0.6440±0.0143 and 0.3665±0.0106 in 3-month-old,1.5-year-old and 3-year-old rabbits respectively,indicating a obvious difference in an age-dependent manner(F=1451.222,P＜0.05).Conclusions The method of culturing LSCs of rabbit in vitro is established in this experiment;LSCs shows a trend of senescence with age;The expression of△Np63 protein in LSCs is associated with age,which suggest that△Np63 protein may play an important role in the aging process of LSCs. |
PDF Full Text Request