Biochemical and Histochemical Investigations on the Pathogenesis of Retinal Degeneration

视网膜变性发病机制的生化和组织化学研究

基本信息

批准号：
60570839
负责人：
UEHARA Fumiyuki
金额：
$ 1.09万
依托单位：
Kagoshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1986
项目状态：
已结题

项目摘要

1. We developed a new technique of isolating cone outer segments using a peanut agglutinin (PNA) adsorbed to a nitrocellulose sheet.2. Histologic changes in the rat neural retina caused by a transscleral administration of lectins into the subretinal space were examined by light microscopy. Ricinus communis agglutinin-1 and concanavalin A induced extensive degenerative changes throughout the neural retina, and on the other hand, wheat germ agglutinin (WGA) involved primarily the photoreceptor layer and the outer nuclear layer. PNA and Dolichos biflorus agglutinin did not produce any change in the neural retina. The primary sites of lectin-binding were detected to be the outer segments of photoreceptors by using HRP-labeled lectins. Triton X-100 extracted fraction of the WGA-induced degenerative retina showed protein bands consisting of newly formed bands (MW 8500 etc).3. We demonstrated that PNA-positive materials exist as well in the cones of rod-free foveal region of the central retina, which indicates a preferential, uniform binding of PNA to cones throughout the retina.4. We develpoed a new procedure to combine the two-dimensional gel electrophoresis and Western blotting followed by lectin reaction, and could detect many more glycoconjugates in the interphotoreceptor matrix than by the conventional method.5. By use of the method described in No 4, glycoconjugates specific for the degenerative retina of C3H mouse were detected. Sialic acid at the terminal of oligosaccharide side-chain of glycoconjugates in the retina might involve the maintenance of normal retina and the degenerative process of the photoreceptor.6. Triton X-100 extracted samples from degenerative retinas (C3H mice) showed a statistically significant reduction of the sialyltransferase activity as compared with normally developed retinas (C57BL; ICR mice).

1. 我们开发了一种利用吸附在硝化纤维片上的花生凝集素（PNA）分离圆锥外节的新技术。2．通过光学显微镜检查经巩膜将凝集素施用到视网膜下空间引起的大鼠神经视网膜的组织学变化。蓖麻凝集素-1 和刀豆球蛋白 A 诱导整个神经视网膜的广泛退行性变化，而另一方面，小麦胚芽凝集素 (WGA) 主要涉及感光层和外核层。 PNA 和 Dolichos biflorus 凝集素没有对神经视网膜产生任何变化。通过使用 HRP 标记的凝集素，检测到凝集素结合的主要位点是光感受器的外部片段。 Triton X-100 提取的 WGA 诱导的退行性视网膜部分显示出由新形成的条带（MW 8500 等）组成的蛋白质条带。 3．我们证明，PNA 阳性物质也存在于视网膜中央凹无视杆细胞区域的视锥细胞中，这表明 PNA 优先、均匀地结合到整个视网膜的视锥细胞中。4．我们开发了一种新的方法，将二维凝胶电泳和蛋白质印迹结合起来，然后进行凝集素反应，并且可以检测到比传统方法更多的光感受器间基质中的糖缀合物。5．通过使用No.4中描述的方法，检测到对C3H小鼠的退化视网膜具有特异性的糖复合物。视网膜糖复合物寡糖侧链末端的唾液酸可能参与正常视网膜的维持和感光细胞的退行性过程。 6． Triton X-100 从退行性视网膜（C3H 小鼠）中提取的样本显示，与正常发育的视网膜（C57BL；ICR 小鼠）相比，唾液酸转移酶活性显着降低。