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 ofp16INK4a gene and Bmi1 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 p16INK4aprotein,Bmi1 protein and Ki67 protein in CECs.Total RNA was extracted from independent corneal sample for the evaluation of p16INK4a mRNA,Bmi1 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/mm2,respectively.Haematoxylin and eosin staining showed the normal structure of corneal epithelium,stroma and endothelium.qRT-PCR examination revealed an age-related increase in p16INK4a mRNA expression in the CECs(F =5.703,P =0.014) and a decrease in Bmi1 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 p16INK4a mRNA in the CECs of the ＞50 years group compared with ＜30 years group and significant decline in Bmil 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 Bmi1 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 Bmi1 gene decreases upon age.These results suggest that p16INK4a gene is associated with senescence of human CECs.
ABSTRACT
The effects of exogenous p16ink4a gene on biological behaviors of human lung cancer cell line with homozygous deletion of p16ink4a gene were investigated. Exogenous p16ink4a gene was transfected by lipofectin into human lung cell line A549, in which p16ink4a gene was homozygously deleted. The expression of pl6ink4a mRNA and protein was detected by RT-PCR and immunocytochemistry, respectively. The changes in the behaviors of the transfected cell lines in vitro and in vivo were observed. In the transfected cell line A549, the exogenous p16ink4a gene could be stably expressed. The growth of A549 cells transfected with p16ink4a gene was obviously slowed down. Flow cytometry revealed that transfection of the exogenous p16ink4a gene resulted in A549 cell lines arrest in G1 phase of cell cycle. The tumorigenicity of these transfected cells in nude mice could be inhibited, and the tumor growth of nude mice was significantly suppressed. It was concluded that exogenous p16ink4a gene may be stably expressed in human lung cancer cell line A549. The expression of the introduced p16ink4a could block lung cancer cells to entry into S phase of cell cycle and inhibit tumor malignant growth both in vitro and in vivo.