RCS大鼠自然病程中视网膜边缘生发区细胞增殖及Shh／Ptc信号途径改变的观察  

Shh/Ptc pathway and retinal stem cells in proliferating marginal region of RCS rats during development of retinitis pigmentosa

作　　者：蹇骞[1] 徐海伟[2] 谢汉平[1] 阴正勤[1] 

机构地区：[1]第三军医大学西南医院全军眼科中心,重庆400038 [2]第三军医大学基础医学部生理学教研室,重庆市神经科学研究所,重庆400038

出　　处：《第三军医大学学报》2009年第11期997-1001,共5页Journal of Third Military Medical University

基　　金：国家重点基础研究发展计划(973计划)(2007CB512203)~~

摘　　要：目的观察遗传性视网膜色素变性RCS大鼠病程发展中视网膜边缘生发区Shh/Ptc信号途径改变及其与该区细胞增殖能力变化的关系。方法RCS大鼠按出生后病程变化分为15、30、60、90 d 4组(免疫组化每组4只,PCR每组3只),以同龄含色素的正常大鼠(Long-Evan’s大鼠)作为正常对照。视网膜组织切片DAPI荧光染色观察外核层(outernuclear layer,ONL)厚度的变化;视网膜细胞增殖标记Ki67免疫荧光染色观察视网膜边缘生发区细胞增殖能力的变化;荧光定量PCR检测视网膜边缘生发区Shh/Ptc信号途径中关键分子Shh、Ptc1、Smo和Gli1 mRNA表达水平。结果①RCS大鼠从第15天开始,随着年龄的增加视网膜ONL逐渐变薄,第90天ONL几乎消失。②正常对照组大鼠视网膜边缘生发区Ki67阳性细胞较少,与同龄正常对照组大鼠相比,出生30、60 d组大鼠Ki67阳性细胞数均显著增多(P<0.05);大鼠各年龄组间比较,60 d组Ki67阳性细胞数明显增多(P<0.01)。③60 d组大鼠视网膜边缘生发区Shh、Ptc1、Smo和Gli1mRNA表达水平明显上调。结论出生后60 d RCS大鼠视网膜边缘生发区Shh/Ptc信号途径的激活,可能刺激了该部位细胞短期的增殖。Objective To observe the relationship of activation of retinal cells in the proliferating marginal region of Royal College of Surgeon＇ s rats （RCS rats, an animal model for inherited retinal degeneration） , and the expressional changes of sonic hedgehog-patched （Shh/Ptc） signaling during development of retinitis pig- mentosa. Methods Totally 28 RCS rats were divided into 4 groups randomly, that is, postnatal day （PND） 15, 30, 60 and 90 group according to the severity of degeneration （in each group, 4 rats for immunofluorescence staining and the left 3 for PCR analysis）. Age-matched Long-Evan＇ s rats without retinal degeneration served as controls. The thickness of outer nuclear layer （ONL） was detected by using DAPI immunofluorescence staining. Ki67, a proliferating marker was detected using immunofluorescence staining, and positive cells were counted per counting-field in the proliferating marginal region of the retinal, mRNA expressions of Shh, Ptc1, Smo and Glil in Shh/Ptc signaling pathway were detected by fluorescent quantitation PCR. Results Compared with age-matched Long-Evan＇ s rats, the thickness of ONL of RCS rats had been decreased with aging since PND 15, and almost disappeared at PND 90 RCS rats. There were more Ki67 positive cells at PND 30 and PND 60 RCS rats than in age-matched Long-Evan＇ s rats （P 〈 0.05） ; compared with PND 15, PND 30 and PND 90 RCS rats, the positive cells at PND 60 RCS rats were increased markedly （ P 〈 0.01 ）. mRNA levels of Shh, Ptc1, Smo and Glil in Shh/Ptc signaling pathway were upregulated at PND 60 RCS rats compared with PND 15, PND 30, PND 90 RCS rats and age-matched Long-Evan＇ s rats. Conclusion Retinal cells are activated to proliferate at the proliferating marginal region within a short time in the retinal of RCS rats at PND 60, which might be regulated by Shh/Ptc pathway.

关 键 词：视网膜色素变性 视网膜干细胞 细胞增殖 shh/Ptc信号途径 荧光定量PCR 

分 类 号：R322.91[医药卫生—人体解剖和组织胚胎学] R329.2[医药卫生—基础医学]