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）吸附在硝化纤维素片上分离锥体外段的新技术。光镜下观察了经巩膜给药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和豆豆蛋白A在整个神经视网膜中引起广泛的退行性改变，另一方面，小麦胚芽凝集素（WGA）主要涉及光感受器层和外核层。PNA和芍药凝集素对神经视网膜无明显影响。利用酶标凝集素检测到凝集素结合的主要位点是光感受器的外段。Triton X-100提取wga诱导的退行性视网膜的部分显示由新形成的条带组成的蛋白质条带（mw8500等）。我们证明PNA阳性物质也存在于视网膜中央无杆中央凹区域的视锥细胞中，这表明PNA在整个视网膜中优先、均匀地结合到视锥细胞上。我们开发了一种新的方法，将二维凝胶电泳与凝集素反应相结合，可以检测到比传统方法更多的糖缀合物。使用第4节描述的方法，检测C3H小鼠退行性视网膜特异性糖缀合物。视网膜糖缀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光感受器的退化过程。Triton X-100从退行性视网膜（C3H小鼠）中提取的样品显示，与正常发育的视网膜（C57BL； ICR小鼠）相比，唾液基转移酶活性有统计学意义的降低。

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).