Keratin 12-deficient mice have fragile corneal epithelia.
Keratin 12-deficient mice have fragile corneal epithelia.
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发表时间：
1996-12
影响因子：
4.4
通讯作者：
W. Kao;Chia-Yang Liu;R. Converse;Atsushi Shiraishi;C. Kao;Masamichi Ishizaki;Thomas Doetschman;John J. Duffy
中科院分区：
文献类型：
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作者：
W. Kao;Chia-Yang Liu;R. Converse;Atsushi Shiraishi;C. Kao;Masamichi Ishizaki;Thomas Doetschman;John J. Duffy
K3-K12角蛋白对的表达是角膜上皮分化的特征。为了阐明角蛋白12在维持角膜上皮完整性中的作用，作者通过基因靶向技术培育角蛋白12缺陷的小鼠。方法在小鼠胚胎干细胞系E14.1中，通过同源重组的方法去除小鼠Krt1.12基因的一个等位基因，并将其与一个DNA构建体进行同源重组，在该DNA构建体中，角蛋白12基因的内含子2和外显子8之间的DNA元件被一个新基因所取代。将同源重组胚胎干细胞注射到小鼠囊胚中，获得嵌合体生殖系。通过立体显微镜、光镜和电镜组织学、Western免疫印迹分析、免疫组织化学、原位杂交和北方杂交对杂合和纯合小鼠的角膜进行临床观察。结果杂合子小鼠（+/-）Krt1.12基因的一个等位基因表现正常，并且不发展任何临床表现（例如，角膜上皮缺损）。纯合子小鼠（-/-）发育正常，角膜上皮轻度糜烂。他们的角膜上皮很脆弱，可以通过轻轻揉眼睛或用微海绵刷来去除。通过免疫组织化学、用表位特异性抗角蛋白12抗体进行的Western免疫印迹分析、用32 P标记的角蛋白12 cDNA进行的北方杂交和用反义角蛋白12核糖探针进行的原位杂交，判断纯合子（-/-）的角膜上皮不表达角蛋白12。光学和电子显微镜显示-/-小鼠角膜上皮的细微异常（即，细胞层数减少）和表面细胞溶解，但基底和基底上细胞中的半桥粒和桥粒数量正常。-/-小鼠角膜上皮细胞基底和基底上角蛋白中间丝数量减少，呈密集束状。这种形态类似于表皮上皮细胞中的角蛋白中间丝，但不同于正常角膜上皮细胞中角蛋白形成细丝状网络的形态。表面上皮细胞缺乏角蛋白中间丝，并且经常从-/-小鼠的角膜表面脱离。结论：角膜特异性K3-K12角蛋白对的存在对于维持角膜上皮的完整性是必不可少的。
PURPOSE Expression of the K3-K12 keratin pair characterizes the corneal epithelial differentiation. To elucidate the role of keratin 12 in the maintenance of corneal epithelium integrity, the authors bred mice deficient in keratin 12 by gene-targeting techniques. METHODS One allele of murine Krt1.12 gene was ablated in the embryonic stem cell line, E14.1, by homologous recombination with a DNA construct in which the DNA element between intron 2 and exon 8 of the keratin 12 gene was replaced by a neo-gene. The homologous recombinant embryonic stem cells were injected to mouse blastocysts, and germ lines of chimeras were obtained. The corneas of heterozygous and homozygous mice were characterized by clinical observations using stereomicroscopy, histology with light and electron microscopy, Western immunoblot analysis, immunohistochemistry, in situ hybridization, and Northern hybridization. RESULTS The heterozygous mice (+/-) one allele of the Krt1.12 gene appear normal and do not develop any clinical manifestations (e.g., corneal epithelial defects). Homozygous mice (-/-) develop normally and suffer mild corneal epithelial erosion. Their corneal epithelia are fragile and can be removed by gentle rubbing of the eyes or brushing with a Microsponge. The corneal epithelium of the homozygote (-/-) does not express keratin 12 as judged by immunohistochemistry, Western immunoblot analysis with epitope-specific anti-keratin 12 antibodies, Northern hybridization with 32P-labeled keratin 12 cDNA, and in situ hybridization with an anti-sense keratin 12 riboprobe. Light and electron microscopy revealed subtle abnormalities in the corneal epithelia of -/- mice (i.e., a decrease in number of cell layers) and cytolysis of superficial cells, but the number of hemidesmosomes and desmosomes are normal in basal and suprabasal cells. The number of keratin intermediate filaments in basal and suprabasal corneal epithelial cells in -/- mice decreases, and they appear as dense bundles. This morphology is similar to that of keratin intermediate filaments in epidermal epithelial, cells but differs from that of normal corneal epithelial cells in which the keratins form fine filamentous networks. The superficial epithelial cells are devoid of keratin intermediate filaments and often detach from the corneal surface of -/- mice. CONCLUSIONS The presence of cornea-specific K3-K12 keratin pairs is essential for the maintenance of corneal epithelium integrity.