Expressions of p16INK4a gene and Bmi1 gene in human corneal endothelial cells of different ages

Abstract

Background p16INK4a gene plays an important role during the aging and senility. So it was well known to be a leading gene associated with aging. Corneal endothelial cells（CECs） always get trapped in the G1 phase due to the lack of proliferative ability. Whether it is relative with cell senescence is unclear. Objective This study was to investigate the expression of p16INK4a gene and Broil gene in human CECs from different aged donors ex vivo. Methods The corneal rims,the residual of corneal tissue preserved in DX solution after penetrating keratoplasty,was used in the present study. Parameters were recorded for the donor, including the age, death to preservation interval and preservation to surgery interval. Corneal endothelium survival rate and endothelial cell density were evaluated by trypan blue-alizarin red dying immediately after penetrating keratoplasty. Routine haematoxylin and eosin staining was also performed to proof the normal structure of the cornea. Sections of corneas from different aged donors were classified into 〈 30 years group, 30-50 years group and 〉 50 years group and were immunostained to assess the expressions of p16INK4a protein, Bmil protein and Ki67 protein in CECs. Total RNA was extracted from independent corneal sampie for the evaluation of p16INK4a mRNA,Bmil mRNA and Ki67 mRNA expression in CECs by quantitative real-time PCR（qRT-PCR）. Results The endothelial cell density of each group was（3069±172） , （2748±64） , （2444 ± 178 ）cells/ram2, respectively. Haematoxylin and eosin staining showed the normal structure of corneal epithelium, stroma and endothelium, qRT-PCR examination revealed an age-related increase in p16~NK4a mRNA expression in the CECs（ F = 5. 703, P = 0. 014 ） and a decrease in Bmil mRNA and Ki67 mRNA expression （ F -- 3. 950 ,P= 0. 042;F--548. 500, P = 0. 000 ）. The further comparison verified a significant elevation in the expression of p16~NK4a mRNA in the CECs of the 〉50 years group compared with 〈30 years group and significant decline in Broil mRNA and Ki67 mRNA the expression （ P = 0. 006,0. 013,0. 000）. Immunohistochemistry in situ confirmed the expression and nuclear localization of p16INK4a protein in CECs, and the expressing intensities of Bmil and Ki67 proteins in the elder donors were weaker than those of the younger donors. The immunofluorescence exhibited that the expressing intensity of p16INK4a protein in CECs of 58 years old donor was higher than that of 23 years old donor, showing a consistent result with that of qRT-PCR. Conclusions Expression of p16INK4a gene increases and that of Bmil gene decreases upon age. These results suggest that p16INK4a gene is associated with senescence of human CECs. Key words: Corneal endothelial cell; Senescence; p16INK4a gene; Broil gene