Song Zhenhua, Wang Ye, Xie Lixin, Zang Xinjie, Yin Hongmei
State Key Lab Cultivation Base, Shandong Provincial Key Lab of Ophthalmology, Shandong Eye Institute, Qingdao, China.
Mol Vis. 2008 Jan 29;14:161-70.
To investigate the expression of p16(INK4a), p21(WAF1/CIP1), p27(KIP1), and p53 in human corneal endothelial cell (HCEC) senescence ex vivo at various donor ages.
Residual corneal tissues obtained after penetrating keratoplasty were used in this study. Age, death-to-preservation interval, and preservation-to-surgery interval of the donors were recorded. Corneal endothelial cell survival and density were evaluated by trypan blue and alizarin red staining immediately after keratoplasty. Fresh frozen sections of donor corneas at various ages (18, 33, 54, and 68 years) were immunostained in situ. Total RNA extracted from age groups of 20, 30, 40, 50, and 60 years was evaluated by reverse-transcriptase polymerase chain reaction (PCR) to reveal the expression of the senescence-related genes, p16(INK4a), p21(WAF1/CIP1), p27(KIP1), and p53, in HCECs. Total RNA extracted from 20-, 24-, 26-, 30-, 50-, 55-, 56-, and 60-year-old donor groups was subjected to real-time PCR analysis for measurement of gene expression. The results of the young (< or =30 years) and the old (> or = 50 years) were compared by the unpaired t-test. Ex vivo senescence of HCECs from the donors at various ages (9, 17, 23, 57, 65, and 67 years) was observed by senescence-associated beta-galactosidase activity (SA-beta-Gal) staining at pH 6.0.
The mean endothelial cell density of the donor corneas was 2,391.4+/-84.6 cells/mm(2), and the survival rate of the endothelial cells was 84.4%+/-5.3%. Hematoxylin and eosin staining showed normal structures of the corneal epithelium, stroma, and endothelium. The expression and nuclear localization of p16(INK4a), p21(WAF1/CIP1), p27(KIP1), and p53 in HCECs were confirmed by immunohistochemistry in situ. Reverse transcriptase PCR examination showed positive target bands of each gene at each age group. An age-related increase in p16(INK4a) expression was observed by real-time PCR (p=0.014). There was no significant difference in the expression levels of p21(WAF1/CIP1), p27(KIP1), and p53 between the young and old donors (p=0.875, 0.472, and 0.430, respectively). Strong SA-beta-Gal activity was observed in the endothelial cells of the old donors while there was weak and little-to-no blue staining in the endothelia from the young.
The population of HCECs exhibiting senescence-like characteristics increases with age. p16(INK4a), p21(WAF1/CIP1), p27(KIP1), and p53 are expressed in HCECs despite donor ages. The p16(INK4a) signal pathway might play a key role in the process of senescence in HCECs.
研究不同供体年龄的人角膜内皮细胞（HCEC）在体外衰老过程中p16（INK4a）、p21（WAF1/CIP1）、p27（KIP1）和p53的表达情况。
本研究使用穿透性角膜移植术后剩余的角膜组织。记录供体的年龄、死亡至保存间隔时间以及保存至手术间隔时间。角膜移植术后立即通过台盼蓝和茜素红染色评估角膜内皮细胞的存活情况和密度。对不同年龄（18、33、54和68岁）供体角膜的新鲜冰冻切片进行原位免疫染色。通过逆转录聚合酶链反应（PCR）评估从20、30、40、50和60岁年龄组提取的总RNA，以揭示HCECs中衰老相关基因p16（INK4a）、p21（WAF1/CIP1）、p27（KIP1）和p53的表达情况。对从20、24、26、30、50、55、56和60岁供体组提取的总RNA进行实时PCR分析以测量基因表达。采用非配对t检验比较年轻（≤30岁）和老年（≥50岁）供体的结果。通过pH 6.0条件下的衰老相关β-半乳糖苷酶活性（SA-β-Gal）染色观察不同年龄（9、17、23、57、65和67岁）供体的HCECs的体外衰老情况。
供体角膜内皮细胞的平均密度为2391.4±84.6个细胞/mm²，内皮细胞的存活率为84.4%±5.3%。苏木精-伊红染色显示角膜上皮、基质和内皮结构正常。通过原位免疫组化证实了p16（INK4a）、p21（WAF1/CIP1）、p27（KIP1）和p53在HCECs中的表达及核定位。逆转录PCR检测显示每个年龄组各基因均有阳性目标条带。通过实时PCR观察到p16（INK4a）表达随年龄增加（p = 0.014）。年轻和老年供体之间p21（WAF1/CIP1）、p27（KIP1）和p53的表达水平无显著差异（分别为p = 0.875、0.472和0.430）。在老年供体的内皮细胞中观察到强烈的SA-β-Gal活性，而年轻供体内皮细胞中的蓝色染色较弱或几乎没有。
表现出衰老样特征的HCECs群体随年龄增加。无论供体年龄如何，p16（INK4a）、p21（WAF1/CIP1）、p27（KIP1）和p53均在HCECs中表达。p16（INK4a）信号通路可能在HCECs衰老过程中起关键作用。
