Research for Anti-Retinal Antibody in Retinal Disorders

已有研究表明，抗视网膜抗体的产生影响了某些视网膜疾病的发病机制。在本项目中，作为第一步，对视网膜疾病患者的血清中是否检测到抗视网膜抗体进行了定性和定量的检测。用视紫红质、视网膜S抗原、组织蛋白酶D和视网膜粗提物作为视网膜抗原。长期广泛的孔源性视网膜脱离以及急性视网膜坏死或炎性色素沉着的视网膜脉络膜旁萎缩的患者对视网膜粗提物的抗体效价较高。克隆了视紫红质、视网膜S抗原、转导蛋白(α-亚基)、IRBP、CRALBP、MEKA、蛋白和33 kDa蛋白的cDNAs，并用合适的质粒载体导入E.ColiJM109，使患者基因组DNA的分子生物学分析能够方便地用于临床部门的实验室检查。此外，为了为未知视网膜抗原的分离做准备，从人脑中提取的mRNA合成了c DNA，并制备了人视网膜c DNA文库。作为本项目的第三步，利用聚合酶链式反应和限制性片段长度多态性技术对视网膜色素变性患者的基因组DNA进行了检测。结果发现1个常染色体显性遗传性视网膜炎家系中的2例视紫红质基因发生点突变，提示视紫红质基因可能与一种视网膜炎的发病机制有关。

It has been suggested that anti-retina antibodies are produced and affect the pathogenesis of some kinds of retinal disorders. In this project, as the first step, it was examined whether or not anti-retina antibodies were detected in the serum of patients with retinal disorders qualitatively and quantitatively. Rhodopsin, retinal S-antigen, cathepsin D and crude extract retina were used as retinal antigens. Patients with prolonged and extensive rhegmatogenous retinal detachment as well as those with acute retinal necrosis or inflammatory pigmented paravenous retinochoroidal atrophy exhibited high titers of antibody to retinal crude extract.As the second step of this project, retina specific antigens were examined. Cloned cDNA's for rhodopsin, retinal S-antigen, transducin (alpha-subunit), IRBP, CRALBP, MEKA, protein and 33kDa protein were obtained and transfected into E. coli JM109 with approproiate plasmid vectors, so that molecular biological analysis of patients' genomic DNA is able to be readily performed as a laboratory examination in a clinical department. Moreover, in order to prepare experiments for the isolation of unknown retinal antigens, cDNA was synthesized from mRNA isolated from human brains and human retinal cDNA library was prepared.As the third step of this project, genomic DNA's from patients with retinitis pigmentosa were examined with a particular interest on the rhodopsin gene using polymerase chain reaction and restriction fragment length polymorphism. As a result, two cases of one pedigree with autosomal dominant retinitis pimentosa was found to have a point mutation in the rhodopsin gene, suggesting that rhodopsin gene could be related to the pathogenesis of a kind of retinitis pimentosa.