Wride M A, Sanders E J
Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Exp Eye Res. 1998 Mar;66(3):371-83. doi: 10.1006/exer.1997.0440.
DNA fragmentation in lens fibre cell nuclei undergoing programmed degeneration was identified by terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick end labelling (TUNEL). Lens epithelial cells in culture were induced to differentiate into lens fibre-like clumps of cells (lentoids) by insulin and it was shown that the TUNEL method was also an effective means of labelling degenerating nuclei in lentoid cells in lens epithelial cell cultures. Using immuno-fluorescence and confocal microscopy, it was shown that TNFalpha and TNF receptor (TNFR1, and TNFR2) immunoreactivity was present in sections of chick embryo lenses. TNFalpha immunoreactivity was associated with the lens epithelium and lens fibres. TNFR1 immunoreactivity was present in lens epithelial cells, cortical lens fibres, and lens fibre cell nuclei, while TNFR2 immunoreactivity had a similar distribution to that of TNFR1, but was not associated with nuclei. Similar patterns of TNFalpha, TNFR1, and TNFR2 immunoreactivity were observed in lens epithelial cell cultures. When added to lens epithelial cell cultures, TNFalpha, at concentrations of 50 to 100 ng ml-1, and agonistic antibodies to both TNFR1 and TNFR2 significantly (P<0.05) enhanced the number of degenerating (TUNEL-positive) nuclei. On the other hand, a neutralising antibody to TNFalpha significantly (P<0. 05) reduced the number of TUNEL-positive nuclei. These results demonstrate that TUNEL is an effective means of labelling degenerating lens fibre nuclei during lens fibre and lentoid differentiation, and suggest a potential role for TNFalpha-like factors and their receptors in the degeneration of lens fibre cell nuclei during lens differentiation. We further suggest that the nuclear degeneration of lens fibre cells is analogous to the nuclear events that occur during apoptosis, and that in lens cells the nuclear degeneration is uncoupled from the plasma membrane events of apoptosis that normally lead to cell death.
通过末端脱氧核苷酸转移酶（TdT）介导的生物素-dUTP缺口末端标记法（TUNEL）鉴定了经历程序性退变的晶状体纤维细胞核中的DNA片段化。培养的晶状体上皮细胞在胰岛素作用下被诱导分化为晶状体纤维样细胞团（类晶状体），结果表明TUNEL法也是标记晶状体上皮细胞培养物中类晶状体细胞退变细胞核的有效方法。利用免疫荧光和共聚焦显微镜观察发现，鸡胚晶状体切片中存在肿瘤坏死因子α（TNFα）和TNF受体（TNFR1和TNFR2）的免疫反应性。TNFα免疫反应性与晶状体上皮和晶状体纤维相关。TNFR1免疫反应性存在于晶状体上皮细胞、皮质晶状体纤维和晶状体纤维细胞核中，而TNFR2免疫反应性的分布与TNFR1相似，但与细胞核无关。在晶状体上皮细胞培养物中也观察到了类似的TNFα、TNFR1和TNFR2免疫反应性模式。当以50至100 ng/ml的浓度添加到晶状体上皮细胞培养物中时，TNFα以及TNFR1和TNFR2的激动性抗体均显著（P<0.05）增加了退变（TUNEL阳性）细胞核的数量。另一方面，TNFα的中和抗体显著（P<0.05）减少了TUNEL阳性细胞核的数量。这些结果表明，TUNEL是在晶状体纤维和类晶状体分化过程中标记退变晶状体纤维细胞核的有效方法，并提示TNFα样因子及其受体在晶状体分化过程中晶状体纤维细胞核退变中可能发挥作用。我们进一步认为，晶状体纤维细胞的核退变类似于凋亡过程中发生的核事件，并且在晶状体细胞中，核退变与通常导致细胞死亡的凋亡质膜事件无关。
