Development of genetically engineered tet HPV16-E6/E7 transduced human corneal epithelial clones having tight regulation of proliferation and normal differentiation
Development of genetically engineered tet HPV16-E6/E7 transduced human corneal epithelial clones having tight regulation of proliferation and normal differentiation
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DOI：
10.1016/s0014-4835(03)00175-1
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发表时间：
2003-10-01
影响因子：
3.4
通讯作者：
Wilson, SE
中科院分区：
文献类型：
--
作者：
Mohan, RR;Possin, DE;Wilson, SE
缺乏人角膜上皮细胞（HCE）的最佳体外模型是研究LICE正常功能和基因调控的主要限制。此外，多层HCE培养物的可用性可以减少在消费品的毒性测试中使用动物。我们已经开发了四环素反应性人乳头瘤病毒（HPV）16-E6/E7转导的HCE克隆，其显示出对增殖和正常分化的严格调节。在Dox存在和不存在（+/-）的情况下，分别通过RNA酶保护试验和蛋白质印迹法检测HPV 16-E6/E7 mRNA和HPV 16-E7和角蛋白K3蛋白的表达。角膜特异性角蛋白k3在+/-Dox中的定位通过免疫细胞化学进行评价。通过细胞计数测量生长因子如肝细胞生长因子（HGF）和表皮生长因子对新鉴定的克隆中的+/-Dox中的细胞增殖的响应。通过光学显微镜和透射电子显微镜评价细胞形态、气液界面多层培养物的形成和超微结构特征。新开发的克隆建立的物理屏障，确定了跨上皮渗透荧光素钠和跨上皮电阻测定在气升分层培养。(C)2003 Elsevier Ltd.保留所有权利。
Lack of an optimal in vitro model of human corneal epithelial (HCE) cells is a major limitation in studying normal functions and gene regulations in LICE. Moreover, availability of a multi-layered HCE culture can reduce the usage of animals in the toxicity testing of consumer products. We have developed tetracycline-responsive human papilloma virus (HPV) 16-E6/E7 transduced HCE clones showing tight regulation of proliferation and normal differentiation. Expression of HPV16-E6/E7 mRNA and HPV16-E7 and keratin K3 proteins was examined by RNase protection assay and western blotting, respectively, in presence and absence (+/-) of Dox in identified clones. Localization of cornea-specific keratin k3 in +/- of Dox was evaluated by immunocytochemistry. The response of growth factors such as hepatocyte growth factor (HGF) and epidermal growth factor to the cellular proliferation in +/- of Dox in the newly identified clones was measured by cell counting. Cellular morphology, formation of multi-layered Cultures at air-liquid interface and ultrastructural features were evaluated by light and transmission electron microscopy. The physical barrier established by the newly developed clones was determined by the transepithelial permeability to sodium fluorescein and transepithelial electrical resistance assays in the airlifted-stratified cultures. (C) 2003 Elsevier Ltd. All rights reserved.