NADPH氧化酶在rd小鼠遗传性视网膜变性中的活化表达

摘要

Background Studies showed that activation of microglia-derived nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in the neurodegenerative diseases and neural cell death in central nervous system.The effect of NADPH on cone degeneration have been determined in rd rats,its role in rod degeneration is relatively less studied.Objective This study was to study the expression of NADPH oxidase in the retinal degenerative process in rd mice and further explore its role in the photoreceptor degeneration.Methods rd Mice at postnatal day 8 (P8),P10,P12,P14,P16 and P18 were collected.The mice were sacrificed,and retinal sections,RNAs and proteins were prepared in above-mentioned time points.The expressions of the gp91 phox,a major subunit of NADPH oxidase,in transcript level and protein level in the retinas were semi-quantitatively detected by real-time PCR and Western blot respectively.Expression of gp91phox was localized in the rd retinas as ageing by immunohistochemstry,and the co-expression of gp91phox with CD11b,a specific marker of microglial cells,was assayed by immunofluorescent double labeling.The C57BL/6N mice were served as controls.The use and care of the animals complied with the Guideline of ARVO.Results Real-time PCR showed that gp91phox mRNA was not expressed in the retinas of C57BL/6N mice.Gp91phox mRNA was found to have less expressed in retinas of P8 rd mice.With aging,the expression level of gp91phox mRNA (gp91phox mRNA/β-actin) in rd mouse retinas was gradually increased with the highest level in P14 mice(1.136±0.370).A significant difference was seen in the gp91 phox mRNA expression among various groups of mice (F=17.81,P =0.00),and gp91phox mRNA expression was significantly elevated in P10,P12,P14,P16 and P18 rd mice compared with P8 rd mice(all at P＜0.05).The expression level of gp91phox protein (A value) in the retinas presented with a similar trend in the rd mice,with a significant difference among the various ages of rd mice and C57BL/6N mice (F =354.00,P＜0.01).The expression level of gp91 phox protein was increased in the rd mice in comparison with the C57BL/6N mice (all at P＜0.05).Immunochemistry revealed that the positive response cells for gp91phox increased in the inner layers of retinas in P10 rd mice and peaked in P14 mice.Immunofluorescent double labeling exhibited that gp91phox were seen to present a co-expression with CD11b,showing an orange fluorescence.Conclusions Expression of NADPH oxidase in the rctinas in the rd mice up-regulates and is parallels to the microglial activation and photoreceptor degeneration,suggesting that NADPH oxidase plays a role in the retinal dystrophy associated with microglial activation.%背景 研究表明,小胶质细胞中烟酰胺二磷酸腺苷(NADPH)氧化酶的活化在中枢神经系统神经元损伤及神经变性疾病中发挥重要作用.已有研究证实NADPH氧化酶在rd小鼠视锥细胞退行性改变中的作用,但关于其在rd小鼠视网膜变性早期视杆细胞病变过程中的作用研究较少. 目的 研究NADPH氧化酶在rd小鼠感光细胞凋亡过程中的活化表达,探讨其在遗传性视网膜变性中的致病作用. 方法 取出生后8、10、12、14、16、18d的rd小鼠各18只,过量麻醉法处死后提取视网膜总RNA和总蛋白,并制备视网膜切片,分别用实时荧光定量PCR及Western blot法测定在rd小鼠感光细胞凋亡过程中视网膜中NADPH氧化酶亚单位gp91phox mRNA及蛋白的定量表达变化;采用免疫组织化学及gp91 phox和CD11b免疫荧光双染法确定gp91 phox在不同鼠龄rd小鼠视网膜中的表达及定位,并与其近交系C57BL/6N小鼠进行比较.结果 实时荧光定量PCR研究证实,C57BL/6N小鼠视网膜中未见gp91 phox mRNA的表达,而生后8d的rd小鼠视网膜中即有少量gp9 1phox mRNA的表达,随着鼠龄的增加,gp91 phox mRNA表达量(gp91phox mRNA/β-actin)逐渐增加,生后14d达到高峰.不同鼠龄rd小鼠视网膜中gp91 phox mRNA表达量差异有统计学意义(F=17.81,P=0.00);生后10、12、14、16、18d的rd小鼠视网膜中gp91phox mRNA表达量均明显高于出生后8d小鼠,差异均有统计学意义(均P＜0.05),以出生后14d表达量最高,为1.136±0.370.随着小鼠鼠龄的增加,视网膜中gp91ph ox蛋白表达量(A值)与其基因表达变化趋势一致,不同鼠龄及C57BL/6N对照小鼠间视网膜中gp91 phox蛋白表达的差异有统计学意义(F=354.00,P＜0.01),不同鼠龄rd小鼠出生后视网膜中gp91 phox蛋白表达量均明显高于C57B L/6N对照小鼠,差异均有统计学意义(均P＜0.05).免疫组织化学法检测表明,rd小鼠出生后10 d视网膜内层gp91 phox阳性细胞开始增多,出生后14d达到高峰,外核层也可见到gp91 phox阳性细胞.免疫荧光双染结果显示,gp91 phox与小胶质细胞标志物CD11b共表达,呈现橙色荧光. 结论 NADPH氧化酶在rd小鼠视网膜中的表达随着鼠龄的增长而增多,其表达与小胶质细胞活化及感光细胞的凋亡相平行,提示NADPH氧化酶可能在小胶质细胞活化导致的感光细胞凋亡中发挥致病作用.