NOX2基因缺陷对rd1小鼠感光细胞凋亡的保护作用

Protection of photoreceptor degeneration in NADPH oxidase 2 deficiency-rd1 mice

目的观察NADPH氧化酶2(NOX2)基因缺陷对遗传性视网膜变性小鼠1(rd1)感光细胞的保护作用。设计实验研究。研究对象出生后14天的NOX2基因缺陷rd1小鼠(实验组)6只及同龄NOX2基因缺陷的C57BL/6N小鼠(对照组1)、无NOX2基因缺陷的rd1小鼠(对照组2)、C57BL/6N野生正常小鼠(对照组3)各6只(共24只)。方法对照组1与对照组2小鼠多次交配获得实验组小鼠并进行基因型鉴定。取该实验鼠及对照组小鼠眼球,对视网膜进行HE染色并测量视网膜外核层厚度,TUNEL染色并计算凋亡细胞占外核层细胞总数百分比、免疫荧光法CD11b抗体标记小胶质细胞并检测NOX2主要亚单位gp91^(phox)蛋白的表达。主要指标视网膜外核层厚度,感光凋亡细胞百分比,gp91^(phox)蛋白的表达量,小胶质细胞活化情况。结果与同龄对照组2相比,实验组小鼠视网膜内外核层排列整齐,其外核层厚度(36.18±2.59)μm明显大于对照组2小鼠(21.45±1.33)μm(t=8.77,P=0.001)。实验组小鼠视网膜凋亡细胞主要出现于外核层,但数量较对照组2明显减少(t=8.46,P<0.001)。与对照组3及对照组1小鼠相比,对照组2小鼠视网膜小胶质细胞明显活化外移浸润外核层,gp91^(phox)表达增加且部分位于小胶质细胞中。而实验组小鼠视网膜gp91^(phox)表达明显减少,小胶质细胞活化受到显著抑制。结论NOX2基因缺陷可有效抑制rd1小鼠视网膜小胶质细胞活化,延缓感光细胞凋亡,有可能成为治疗遗传性视网膜变性疾病的潜在靶点。

Objective To observe the delay of photoreceptor degeneration in the NADPH oxidase2(NOX2)deficiency-rdl mice.Design Experiment study.Participants 6 NOX2-deficiency rd1 mice(experimental group)at postnatal 14d and their control groups at same age,including 6 NOX2-deficiency C57BL/6N mice(control group 1),6 rdl mice without NOX2-deficiency(control group 2)and 6 C57BL/6N mice(control group 3)were included.Methods The experimental group were obtained and appraised through repeated mating of control group 1 and control group 2 mice.The thickness of outer nuclear layer(ONL)was measured on HE staining.Photoreceptor apoptosis was examined by TUNEL assay and the percentage of apoptotic cells in total cells of ONL was counted.The expression of gp91^(phox),a major subunit of NOX2,as well as activation of microglial cells were studied by immunofluorescence and double-labeling technique.Main Outcome Measures Thickness of outer nuclear layer;percentage of TUNEL positive cells;expression of gp91^(phox);microglial activation.Results In the experimental group,the thickness of outer nuclear layer was 36.18±2.59μm,which was significantly higher than that of control group 2(21.45±1.33μm,t=8.77,P=0.001).TUNEL positive retinal apoptotic cells,which mainly appeared in the ONL of experimental group,were markedly reduced(t=8.46,P<0.001)when compared with aged matched control group 2.Compared with control group 3 and control group 1,microglial cells were markedly activated in the retina of control group 2 and infiltrated ONL.Expression of gp91^(phox) was also increased in part of microglial cells.In contrast,the experimental group showed dramatically down-regulated expression of both gp91^(phox) and activation of microglial cells.Conclusion Deficiency of NOX2 gene effectively delayed photoreceptor cell loss in the rdl mice possibly through inhibition of microglial cells.NOX2 may become a new and potential therapeutic target for inherited retinal degeneration.