Gropp K E, Szél A, Huang J C, Acland G M, Farber D B, Aguirre G D
James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Exp Eye Res. 1996 Sep;63(3):285-96. doi: 10.1006/exer.1996.0117.
We have used immunocytochemistry and in situ hybridization to examine the expression of photoreceptor specific genes in retinas of normal dogs and those affected with hereditary cone degeneration (cd), a rare autosomal recessive disorder that selectively affects cones. In the cd retina, cone disease begins early in life; cones are lost by extrusion of the nucleus into the inner segment, and later by displacement of the nucleus, surrounded by a thin rim of cytoplasm, into the interphotoreceptor space. Two micrometer sections from the superior and inferior retinal meridians, extending from the optic disk to the ora serrata, were used for in situ hybridization with a bovine rod opsin and human red/green cone opsin cRNA probes, or were reacted with antibodies directed against photoreceptor-specific proteins and visualized with appropriate biotinylated antibodies. Antibodies against the following proteins were used: alpha- and beta 3-transducins, phosducin, alpha/beta- and gamma-phosphodiesterases, COS-1, and OS-2, opsin, S-antigen and IRBP. Immunoreactivity or hybridization labeling was evaluated in unstained sections; cone pathology was judged in adjacent Toluidine Blue-stained sections. With these methods it was possible to evaluate immunoreactivity or hybridization labeling and cone pathology at the single cell level. Both middle-(COS-1) and short-(OS-2) wavelength-sensitive cones were present in controls and cd affected retinae at 2.2 months, and distinct transcripts of the red/green cone pigment gene were identified in the majority of cones in both normal and affected retinas at this age. However, beta 3-transducin immunoreactivity was completely absent from cd-affected cone outer segments. Both cone types were present but in reduced numbers in older animals (11.5 and 17 months), and no reactivity to beta 3-transducin was noted. No differences were found with the other antibodies used. The specific absence of beta 3-transducin immunoreactivity from the cone outer segments suggests a potential involvement of the beta 3-transducin gene or gene product in the disease process.
我们运用免疫细胞化学和原位杂交技术，检测正常犬以及患有遗传性视锥细胞变性（cd）的犬视网膜中光感受器特异性基因的表达情况。遗传性视锥细胞变性是一种罕见的常染色体隐性疾病，选择性地影响视锥细胞。在患有视锥细胞变性的视网膜中，视锥细胞疾病在生命早期就开始出现；视锥细胞核先挤入内节，随后细胞核连同周围薄薄一层细胞质一起移位到光感受器间腔，导致视锥细胞丢失。从视盘延伸至锯齿缘的视网膜上下子午线处取2微米厚的切片，用于与牛视杆视蛋白和人红/绿视锥视蛋白cRNA探针进行原位杂交，或与针对光感受器特异性蛋白的抗体反应，并用适当的生物素化抗体进行可视化观察。使用了针对以下蛋白质的抗体：α和β3 -转导蛋白、磷转导蛋白、α/β -和γ -磷酸二酯酶、COS - 1、OS - 2、视蛋白、S -抗原和IRBP。在未染色的切片中评估免疫反应性或杂交标记；在相邻的甲苯胺蓝染色切片中判断视锥细胞病变情况。通过这些方法，可以在单细胞水平评估免疫反应性或杂交标记以及视锥细胞病变。在2.2个月大时，对照组和患有视锥细胞变性的视网膜中均存在中波长敏感（COS - 1）和短波长敏感（OS - 2）视锥细胞，并且在这个年龄段，正常和患病视网膜的大多数视锥细胞中都鉴定出了红/绿视锥色素基因的独特转录本。然而，在患有视锥细胞变性的视锥细胞外节中，β3 -转导蛋白免疫反应性完全缺失。在年龄较大的动物（11.5个月和17个月）中，两种视锥细胞类型均存在，但数量减少，且未观察到对β3 -转导蛋白的反应性。使用的其他抗体未发现差异。视锥细胞外节中β3 -转导蛋白免疫反应性的特异性缺失表明β3 -转导蛋白基因或基因产物可能参与了疾病进程。
