RCS变性大鼠感光细胞凋亡及其视网膜电图与caspase-9时空表达的关系研究

目的：研究caspase-9在英国皇家外科学院(RCS)变性大鼠视网膜的时空表达状况与其视网膜电图(ERG)以及感光细胞凋亡的关系，以探讨caspase-9在感光细胞凋亡中的作用。 方法： 不同日龄RCS变性大鼠，检查ERG后处死取视网膜行caspase-9的免疫组化、荧光活力分析、Western blotting及凋亡TUNEL检测。 结果： 15及20 d变性大鼠ERG b波潜伏期分别为(58.60±3.42)ms、(56.45±1.08)ms，振幅分别为(109.07±14.50)mV、(109.00±7.35)mV，两组间无显著性差异，25和30d组接近熄灭型。TUNEL阳性细胞只在变性大鼠的外颗粒层表达，25 d最明显。25 d后的变性大鼠感光细胞内节可见明显caspase-9阳性表达，其余各组未见表达。20 d caspase-9表现出最大活力［(10.98±0.13)nmol·g-1·min-1］。Western blotting分析显示caspase-9裂解条带在20 d时最明显，15 d组和对照组不表达。 结论： caspase-9的表达与视网膜感光细胞凋亡、视功能丧失存在时空一致性。

Relation between the time-spatiotemporal expression of caspase-9 and photoreceptor apoptosis as well as electroretinography in RCS rats

AIM: To investigate the relation between the time- spatiotemporal expression of caspase-9 and photoreceptor apoptosis as well as electroretinography (ERG) in Royal College of Surgeons (RCS) rats. METHODS: Twelve non-pigmented degenerative RCS rats at age of 15, 20, 25, 30 days were sacrificed after ERG examination. The caspase-9 expression was analyzed with immunohistochemical staining, fluoro-metric activity assay and Western blotting. Apoptotic retinal neurons were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. RESULTS: The latency and amplitude of b-wave were (58.60±3.42) ms and (109.07±14.50) mV, (56.45±1.08) ms and (109.00±7.35) mV, respectively, in 15 and 20 days old degenerative RCS rats. The ERG resembled flat form in the degenerative RCS rats at age of 25 and 30 days. TUNEL-positive cells were located only in the outer nuclear layer, mostly prevailed in 25-day-old group. Caspase-9 protein expressed in the rat retina started to be found immunohistochemically in 25-day-old and was most predominant in the inner segment. Fluorometric activity assay showed that caspase-9 activity reached its peak, (10.98±0.13) nmol·g-1·min-1 at 20th day. Western blot assay revealed the small cleavage fragmentation at 20th day. No cleavage fragmentation was revealed in the 15-day-old degenerative RCS rats and all other control rats. CONCLUSION: The activation and time-spatiotemporal expression of caspase-9 are coincidence with photoreceptor apoptosis as well as the loss of vision function in RCS degenerative rats, which implicates the important role of caspase-9 in photoreceptor apoptosis.