硫酸软骨素蛋白多糖在视网膜变性大鼠中的表达

摘要

目的 研究硫酸软骨素蛋白多糖(CSPGs)在碘酸钠(NaIO3)诱导的视网膜色素上皮(RPE)细胞变性大鼠中的表达情况.方法 实验研究.24只Sprauge-Dawley(SD)大鼠随机分为4组,正常对照组、造模7d组、造模14 d组、造模28 d组,每组6只.造模组腹腔注射3％ NaIO3(100 mg/kg);取眼球做病理检查,苏木素-伊红(HE)染色及细胞凋亡检测,验证视网膜变性动物模型的建立;免疫荧光法观察视网膜变性大鼠视网膜上CSPGs表达的时空规律；逆转录-聚合酶链反应(RT-PCR)法检测CSPGs多功能蛋白聚糖(Versican) mRNA的表达情况；免疫组织化学法观察视网膜上小胶质细胞表达的巨噬细胞特异性抗原CD68的分布特点.多组比较采用单因素方差分析,两样本间比较采用独立样本t检验.结果 注射NaIO3后,模型大鼠视网膜出现变性改变,且光感受器发牛渐进性凋亡,造模7、14、28 d组凋亡率分别为(21.0±3.5)％、(32.3±2.3)％、(41.7±2.6)％,各组间差异有统计学意义(F=205.27,P＜0.01).造模7d组,CS-56、CD68在脉络膜、视锥视杆层、内核层、节细胞层表达;造模14 d组,CS-56、CD68进一步出现在外核层;造模28 d组,CSPGs继续迁移到外丛状层.随造模时间延长,各层荧光表达逐渐增强,CD68在视网膜各层的表达也明显增多,强度分别为:1.33±0.52、2.67±0.82、4.00±1.10和7.17±1.33,各组间差异有统计学意义(F=38.45,P＜0.01).造模组Versican mRNA表达(1.02±0.06)显著高于正常对照组(0.23±0.02),差异有统计学意义(t=-26.05,P＜0.01).结论 在碘酸钠诱导的视网膜变性动物模型中,随时间延长CSPGs表达范围扩大,表达量增加,并与小胶质细胞分布在大致相同区域,提示CSPGs可能来源于小胶质细胞.%Objective To study the expression of chondroitin sulfate proteoglycans (CSPGs) in retinal pigment epithelium cell degeneration rat induced by sodium iodate (NaIO3) intraperitoneal injection.Methods In this experimental study,a total of 24 Sprague-Dawley (SD) rats were used.And divided into control group,7 days group,14 days group and 28 days group,with 6 rats in each group.NaIO3 (3％,100 mg/kg) was intraperitoneally injected in 18 rats to establish the retinal degeneration models.HE and Tunel assay were performed to evaluate the retinal degeneration.The expression of CSPGs in rat retinas was detected by immunofluorescence; the expression of Versican mRNA was analyzed by retrovirus-polymerase chain reaction (RT-PCR).The macrophage-specific antigen CD68,secreted by microglia,was detected by an immunohistochemical method.The data was statistically analyzed by one-way ANOVA and independent samples t test.Results HE showed morphological retinal degeneration in rats after NaIO3 injection.The apoptosis of photoreceptors appeared more severe with the development of retinal degeneration in rat retinas after NaIO3 injection.The apoptosis rates were (21.0±3.5)％,(323±2.3)％ and (41.7±2.6)％ in 7 days.14 days and 28 days groups,respectively.after NaIO3 injection,with statistically significant differences between each group (F=205.27,P＜0.01).CS-56 and CD68 appeared in the choroid,photoreceptor outer segment debris zone (DZ),the internuclear layer (INL) and the ganglion cell layer (GCL) 7 days after NaIO3 injection and spread to the outer nuclear layer (ONL) 14 days after NaIO3 injection.Twenty-eight days after NaIO3 injection,CSPGs continued to expand to the outer plexiform layers (OPL).With the passage of time after NaIO3 injection,CS-56 fluorescence and CD68 intensity became stronger in each layer.CD68 intensity was 1.33±0.52,2.67±0.82,4.00±1.10 and 7.17±1.33 in normal rat retinas,7 days,14 days and 28 days after NaIO3 injection,and there were statistically significant differences between each group (F=38.45,P＜0.01).The expression of Versican mRNA,the N-terminal fragment of CSPGs,in the retinal degeneration animals was 1.02±0.06,which was significantly higher than in the control rats (0.23±0.02) (t=-26.05,P＜0.01).Conclusion With the passage of time after NaIO3 injection,the range of CSPGs continued to expand and the expression intensity became stronger in rat retinas.The same distribution of CSPGs and microglia suggested that microglia constitute a source of CSPGs in the degenerating rat retina.