小鼠光感受器细胞丢失环境对内在光感受性视网膜神经节细胞的影响

THE EFFECT OF CLASSICAL PHOTORECEPTOR DEGENERATION ON INNER PHOTOSENSITIVE RETINAL GANGLION CELLS IN RD MICE RETINA

目的研究视网膜变性小鼠(rd)光感受器细胞丢失病变过程中内在光感受性视网膜神经节细胞(inner photosensitive retinal ganglion cells,ipRGCs)及其黑视素表达的变化。方法取出生后1月龄、3月龄和6月龄视网膜变性小鼠和正常对照小鼠,分离视网膜行melanopsin免疫组化染色,阳性细胞计数;应用荧光定量PCR和western-blot,检测不同病变阶段rd小鼠视网膜中melanopsin基因和蛋白的表达情况,分析组间差异。结果正常小鼠视网膜约1%视网膜神经节细胞呈melanopsin阳性表达,分布于全视网膜。rd小鼠melanopsin阳性神经节细胞数量同正常小鼠相比,各年龄组均无明显差异;荧光定量PCR结果显示,与正常对照组和1月龄rd小鼠组相比,3月龄和6月龄rd小鼠视网膜melanopsin mRNA的表达量显著上调,且差异有统计学意义(P<0.05)。western-blot结果显示,随着出生年龄的增长rd小鼠视网膜melanopsin蛋白表达量呈上升趋势,6月龄rd小鼠与1月龄rd小鼠间差异有统计学意义(P<0.05)。结论视网膜变性小鼠病变进程中,内在光感受器视网膜神经节细胞数量无明显变化,而基因和蛋白分析显示melanopsin表达逐渐上调,在病变晚期尤为显著。提示在视网膜变性微环境中,经典光感受器细胞丢失后,ipRGCs作为第三类光感受器细胞可能试图代偿感光功能,上调特异感光色素的表达。

Objective To explore the effect of photoreceptor degeneration on inner photosensitive retinal ganglion cells and its melanopsin expression in rd mice retina. Methods The retinas of postnatal 1 month, 3- month and 6- month-old rdmice and control mice were separated and immunohistochemistry stained, the number of melanopsin - positive ganglion cells was counted. The expression of metanopsin mRNA and proteinof rdmice retina at different disease stages were investigated by real- time PCR and western- blot, the difference was analyzed. Results About 1 % of retinal ganglion cells was melanopsin- positive and distributed in the whole retina in normal mice. Compared with normal mice, the number of melanopsin- positive ganglion cells in all rd mice group was not significantly different. Real - time PCR results showed that, compared with normal control mice and 1 - month - old rd mice group, the expression of melanopsin mRNA of rdmice retina were significantly increased, and gradually increased with age. The western - blot showed that, the melanopsin protein expression showed an upward trend with age in rd mice, and there was significantly difference of the expression between 6 months and 1 - month-old rd mice. Conclusions The number of inner photosensitive retinal ganglion cells is not significant changed during the photoreceptor degeneration in rd mice. But the melanopsin expression is gradually increased both of gene and protein,particularly significant in late stage of disease. The results imply that, as the third type of photoreceptor, the ipRGCs increases the expression of specific light- sensitive pigment- melanopsinafter the classical photoreceptor loss,may be in order to compensatory photoreceptive function.