Localisation of Notch and Delta in the Human Cornea
Localisation of Notch and Delta in the Human Cornea
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DOI：
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发表时间：
2003-05
影响因子：
4.4
通讯作者：
J. Albon;Aihua Ma;J. Cai;M. Boulton
中科院分区：
文献类型：
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作者：
J. Albon;Aihua Ma;J. Cai;M. Boulton
目的：研究在各种上皮细胞的细胞命运决定中起重要作用的Notch及其配体Delta是否存在于人角膜中。方法：使用针对 Notch 1 和 Delta (AutogenBioclear) 的抗体对尸体 24 小时内取出的人角膜冷冻切片进行免疫荧光标记，然后在适当的 Alexor Fluor (Molecular Probes) 二抗中孵育。对分离的角膜上皮细胞、角膜细胞和内皮细胞进行 SDS-PAGE 和蛋白质印迹以确认定位模式。结果：Notch 的免疫荧光定位在所有基底上细胞的整个角膜上皮中得到证实，即在翼和浅表上皮细胞的边界、中央、外周和角膜缘区域。在任何区域的上皮基底细胞层中均未检测到标记。相比之下，δ似乎在角膜上皮的所有区域和所有细胞层的细胞边界中表达，并且周边基底细胞下方的强度增加。角膜角膜细胞和内皮细胞中均不存在切迹的免疫标记，但在角膜基质内的少数角膜细胞中观察到δ的定位。免疫印迹证实了上述细胞定位：80kDa Delta 蛋白存在于 Triton 可溶的角膜上皮细胞部分中。结论：角膜上皮中Notch和Delta的存在表明它通过调节细胞命运和分化在角膜稳态中发挥关键作用。
Purpose: To investigate if Notch and its ligand Delta, which play important roles in cell fate decisions in various epithelia, are present in the human cornea. Methods: Cryosections of human corneas, retrieved within 24 hours cadaver time, were immunofluorescently labelled using antibodies directed against Notch 1 and Delta (AutogenBioclear) followed by incubation in appropriate Alexor Fluor (Molecular Probes) secondary antibodies. SDS-PAGE and Western blotting of isolated corneal epithelial cells, keratocytes and endothelial cells were performed to confirm the localisation pattern. Results: Immunofluorescent localisation of Notch was demonstrated throughout the corneal epithelium in all suprabasal cells i.e. in the borders of the wing and superficial epithelial cells, in central, peripheral and limbal regions. Labelling was not detected in the epithelial basal cell layer in any region. In contrast, delta appeared to be expressed in cell borders throughout all regions, and all cell layers of the corneal epithelium, with an increased intensity underlying peripheral basal cells. Immunolabelling for notch was absent in both corneal keratocytes and the endothelium, however localisation of delta was observed in a small number of keratocytes within the corneal stroma. Immunoblotting confirmed the aforementioned cellular localisation: the 80kDa Delta protein was present in the Triton-soluble corneal epithelial cell fraction. Conclusions: The presence of Notch and Delta in the corneal epithelium suggests that it plays a pivotal role in corneal homeostasis by regulating cell fate and differentiation.