Biochemical and Histochemical Investigations on the Pathogenesis of Retinal Degeneration

1.我们开发了一种利用花生凝集素(PNA)吸附在硝酸纤维素膜上来分离锥体外节的新技术。用光学显微镜观察了经巩膜向视网膜下间隙注射凝集素引起的大鼠神经视网膜的组织学变化。蓖麻凝集素-1和刀豆蛋白A在整个神经视网膜引起广泛的变性改变，另一方面，小麦胚凝集素(WGA)主要涉及光感受器层和外核层。PNA和两花扁豆凝集素对神经视网膜没有任何影响。用HRP标记的凝集素发现，凝集素结合的主要部位是光感受器的外节段。Triton X-100提取的WGA诱导的变性视网膜蛋白条带由新形成的条带组成(分子量为8500等)。我们证明PNA阳性物质也存在于视网膜中央无杆中心凹区域的视锥细胞中，这表明PNA与整个视网膜的视锥细胞具有优先的、均匀的结合。我们开发了一种新的方法，将双向凝胶电泳和Western blotting结合起来，然后进行凝集素反应，可以检测到比传统方法更多的光感受器基质中的糖结合物。用文献[4]中所述的方法，检测了C3H型小鼠视网膜变性的特异性糖偶联物。视网膜糖结合物寡糖侧链末端的唾液酸可能参与了正常视网膜的维持和光感受器的退变过程。从变性视网膜(C3H小鼠)提取的Triton X-100样品显示，与正常发育的视网膜(C57BL；ICR小鼠)相比，唾液酸转移酶活性显著降低。

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).