中文摘要：

背景p16^INK4a基因在细胞老化和早衰过程中发挥着重要作用，是目前已知的细胞衰老的主导基因。角膜内皮细胞（CECs）周期停滞于G。期且体内缺乏增生能力，是否与细胞衰老有关尚未得知。目的探讨不同年龄的正常人CECs中p16^INK4a基因及其抑制基因Bmil的表达。方法收集临床上DX液保存的行角膜移植手术后剩余的供体周边环状角膜组织，记录供体年龄。经锥虫蓝一茜素红双染法观察CECs的活性；行苏木精一伊红染色以证实角膜材料的组织结构正常。将收集到的角膜环材料按照年龄分为〈30岁组、30～50岁组和〉50岁组，每组30个角膜环，其中15个用于总RNA提取，另外15个角膜环等分为3份，分别用于免疫组织化学检测、免疫荧光检测和CECs活性的观察。提取每个角膜环的总RNA，行实时荧光定量PCR检测，观察p16^INK4amRNA、BmilmRNA和Ki67mRNA在CECs中的表达。行冰冻切片免疫组织化学染色，检测p16^INK4a、Bmil和Ki67蛋白在CECs中的表达。免疫荧光法检测p16”“。在CECs的表达。结果苏木精一伊红染色结果显示，供体角膜上皮、基质和内皮结构完整，排列规则。实时荧光定量PCR检测显示随年龄增长，p16^INK4a。mRNA的表达增强，而BmilmRNA和Ki67mRNA的表达减弱，差异均有统计学意义（F=5．703，P=0．014；F=3．950，P=0．042；F=548．500，P=0．000），其中与〈30岁组比较，〉50岁组p16^INK4amRNA在CECs中的表达量明显升高，而BmilmRNA和Ki67mRNA的表达均明显降低，差异均有统计学意义（P=0．006、0．013、0．000）。免疫组织化学结果可见，p16^INK4a。蛋白在各组CECs中均有表达，主要位于细胞核内，而Bmil与Ki67在年老供体的表达强度均弱于年轻供体，与实时荧光定量PCR的结果基本一致。免疫荧光染色显示，年老供体CECs p16^INK4a的表达强度强于年轻供体。结论细胞衰老主导基因p16^INK4a的表达随?

英文摘要：

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 d