Molecular biological study of corneal dystrophy

为了获得角膜营养不良的候选基因，使用角膜和虹膜RNA作为探针，对来自冈山大学医学院分子生物学和生物化学系的兔角膜cDNA文库进行加/减筛选。分离出对应于三个铁蛋白H-链、NADH-泛醌氧化还原酶B22亚基、α 1 VIII型胶原、25KDa FKBP-506结合蛋白（FKBP 25）、血小板反应蛋白2的十三个克隆和六个未知克隆。RT-PCR分析表明FKBP 12和25在兔角膜、视网膜、大脑、小脑中有高表达。在培养的角膜上皮细胞中也有高表达。虽然从这些分离的mRNA转运的蛋白质不是角膜特异性的，但它们将在角膜中发挥重要作用。没有一个分离的已知mRNA不定位于染色体5、16或20。我们计划进一步的研究，包括利用BAC筛选和FISH技术对未知克隆进行染色体定位，并从文库中随机抽取约700个克隆，进行部分测序和互联网同源性检索，由于角膜营养不良的诊断需要确认，我们对159个角膜纽扣样本进行了病理学评价。我们对角膜营养不良进行了细致的诊断，发现了Avellino角膜营养不良，这在日本尚属首次报道。我们从这些病人身上提取了DNA，并将其储存起来进行进一步分析。我们还完成了角膜组织基因转移基础研究的准备工作。

To obtain the candidate genes for the corneal dystrophies, plus/minus screening of the rabbit corneal cDNA library from the Department of Molecular Biology and Biochemistry, Okayama University Medical School was performed using corneal and iris RNA as probes. Thirteen clones corresponding to three ferritin H-chain, a NADH-ubiquinone oxidoreductase B22 subunit, an alpha 1 type VIII collagen, a 25KDa FKBP-506 binding protein (FKBP25), a thrombospondin 2 and six unknown clones were isolated. RT-PCR analysis showed that FKBP 12 and 25 highly express in the rabbit cornea, retina cerebrum, cerebellum. They also highly express in the cultured corneal epithelial cells. Although proteins tranlocated from these isolated mRNA are not corneal specific, they will play an important role in the cornea. None of the isolated known mRNAs does not map to the chromosome 5,16 or 20. We plan the further study including the chromosomal localization of the unknown clones using the BAC screening and FISH.We also randomly extracted the about 700 clones from the library, sequenced partially and done the homology search using the Internet service.Since the confirmation of the diagnosis of the corneal dystrophies was required, we evaluated the 159 samples of the corneal button pathologically. We elaborately diagnosed the corneal dystrophies and found the Avellino corneal dystrophy which is the first report in Japan. We extracted the DNA from these patients and stocked them for the further analysis. We also completed the preparation for the basic study for the gene transfer to the corneal tissue.