Egan C A, Savre-Train I, Shay J W, Wilson S E, Bourne W M
Department of Ophthalmology, Mayo Clinic Rochester, Minnesota 55905, USA.
Invest Ophthalmol Vis Sci. 1998 Mar;39(3):648-53.
To investigate the telomere hypothesis of cellular aging as the mechanism for cell cycle arrest in normal human corneal endothelium.
The corneal endothelium and epithelium from 21 human corneas from 13 donors 5 weeks to 84 years of age were dissected and frozen at -70 degrees C. Purified DNA, digested with the restriction enzyme, HinfI, was run on 0.7% agarose gels, probed with radiolabeled (AATCCC)4, and exposed to a phosphor screen. The length of the terminal restriction fragment (TRF) was determined by densitometry.
The cells of the corneal endothelium had TRF lengths ranging from 11.0 to 14.0 kbp (mean, 12.2 +/- 0.9). Corneal epithelial specimens showed TRF lengths that were always less than (mean, 10.4 +/- 1.0; range 9.0-12.0) the corresponding endothelial TRF lengths. Human corneal endothelial cells, transformed with human papillomavirus type 16 oncogenes E6 and E7, showed decreasing TRF lengths from 11 kbp at population doubling level (PDL) 15 to 9.5 kbp at PDL 73. Neither the endothelial and epithelial cells from human donors nor the transformed pre-immortalized human endothelial cells showed evidence of telomerase activity.
Human corneal endothelial cells have long telomeres throughout life. Their limited replicative ability does not appear to result from critically short telomere lengths.
研究细胞衰老的端粒假说，作为正常人角膜内皮细胞周期停滞的机制。
从13名年龄在5周至84岁的供体获取21个人类角膜，分离出角膜内皮和上皮组织，并在-70℃下冷冻。用限制性内切酶HinfI消化纯化的DNA，在0.7%琼脂糖凝胶上进行电泳，用放射性标记的(AATCCC)4探针杂交，并与磷屏曝光。通过密度测定法确定末端限制片段(TRF)的长度。
角膜内皮细胞的TRF长度范围为11.0至14.0 kbp(平均值为12.2±0.9)。角膜上皮标本显示的TRF长度总是小于相应内皮细胞的TRF长度(平均值为10.4±1.0；范围为9.0 - 12.0)。用人乳头瘤病毒16型癌基因E6和E7转化的人角膜内皮细胞，其TRF长度从群体倍增水平(PDL)15时的11 kbp下降到PDL 73时的9.5 kbp。来自人类供体的内皮细胞和上皮细胞以及转化的前永生化人内皮细胞均未显示端粒酶活性的证据。
人角膜内皮细胞一生都有较长的端粒。其有限的复制能力似乎并非源于端粒长度严重缩短。
