目的探讨形觉剥夺性近视(FDM)豚鼠视网膜中m6A去甲基化酶AlkB同源蛋白5(ALKBH5)表达变化及其参与近视的作用机制。
方法采用随机数字表法将30只健康SPF级3周龄三色豚鼠分为正常对照组和实验组，每组15只。实验组中眼罩遮盖右眼作为FDM组，暴露左眼为自身对照组。分别于实验前及实验1、2、3和4周时进行豚鼠眼生物学参数测量。采用带状光检影镜测量屈光度，采用A型超声仪测量眼轴长度。实验4周，通过免疫组织化学染色和免疫荧光染色检测ALKBH5在豚鼠视网膜中的表达分布。采用实时荧光定量PCR和Western blot法检测豚鼠视网膜中ALKBH5 mRNA和蛋白表达情况。
结果与正常对照组和自身对照组相比，实验2、3和4周，FDM组豚鼠近视屈光度明显增加，眼轴显著增长，差异均有统计学意义(均 P<0.001)。免疫组织化学染色和免疫荧光染色显示，ALKBH5分布在视网膜神经纤维层、视锥视杆细胞层和视网膜色素上皮(RPE)层，其中以神经纤维层和RPE层为主。正常对照组、自身对照组和FDM组豚鼠ALKBH5蛋白相对荧光强度值分别为1.000±0.204、0.874±0.076和0.571±0.053，FDM组视网膜中ALKBH5蛋白荧光强度值明显小于正常对照组和自身对照组，差异均有统计学意义( t＝4.069， P＝0.006； t＝5.176， P＝0.014)。造模后4周，FDM组豚鼠视网膜中ALKBH5 mRNA和蛋白相对表达量明显低于正常对照组和自身对照组，差异均有统计学意义(均 P<0.01)。
结论FDM组豚鼠视网膜中m6A去甲基化酶ALKBH5表达下降，ALKBH5及相关m6A甲基化修饰可能参与了近视的发生和发展。
ObjectiveTo investigate the alteration of m6A demethylase AlkB homolog 5 (ALKBH5) expression and its impact on form-deprivation myopia (FDM) retina in guinea pigs.
MethodsThirty normal SPF grade 3-week-old tricolor guinea pigs were randomly divided into normal control group and experimental group, with 15 in each group.In the experimental group, the right eyes were covered as FDM group and the left eyes uncovered were set as self-control group.Ocular biometry was performed at one-week intervals from baseline to week 4 of the experiment.Spherical equivalent was detected by streak retinoscopy and axial length was measured by A-scan ultrasonography.Animals were sacrificed after 4 weeks of modeling.The distribution and expression of ALKBH5 protein in the guinea pig retina was detected by immunohistochemical and immunofluorescence staining.Expression of ALKBH5 mRNA and protein in guinea pig retina was detected by real-time fluorescence quantitative PCR and Western blot, respectively.The use of animals in ophthalmic and vision research followed the tenets of Animal Research in Vision and Ophthalmology, and the study was approved by the Ethics Committee of North Sichuan Medical College (No.2023087).
ResultsAt weeks 2, 3, and 4 after myopia induction, diopters and axial lengths were significantly higher in the FDM group than in the normal control group and the self-control group (all at P<0.001). Immunohistochemistry and immunofluorescence assays showed that ALKBH5 protein was expressed in the retinal nerve fiber layer, rod/cone photoreceptor cells, and retinal pigment epithelium (RPE) layer, and was highly expressed in the retinal nerve fiber layer and RPE layer.The relative ALKBH5 immunofluorescence intensity in the normal control group, self-control group and FDM group was 1.000±0.204, 0.874±0.076 and 0.571±0.053, respectively, which was lower in the FDM group than in the normal control and self-control groups, showing statistically significant differences ( t=4.069, P=0.006; t=5.176, P=0.014). After 4 weeks of modeling, ALKBH5 mRNA and protein expressions were significantly lower in FDM group than in normal control and self-control groups (both at P<0.01).
ConclusionsThe expression of m6A demethylase ALKBH5 is decreased in the retina of FDM guinea pigs, suggesting that ALKBH5 and related m6A methylation modification may be involved in the development and progression of myopia.
向小玲,廖萱,孟茄玉,等. m6A去甲基化酶ALKBH5在形觉剥夺性近视豚鼠中的表达变化及其意义[J]. 中华实验眼科杂志,2024,42(04)：315-321.
DOI:10.3760/cma.j.cn115989-20231028-00156版权归中华医学会所有。
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向小玲：参与研究设计和实施、收集数据、资料分析及解释、文章撰写及修改；廖萱：参与研究设计、文章修改、对文章的知识性内容作批判性审阅及定稿；孟茄玉：分析/解释数据、统计分析；周桂梅、周红、周羡媚：采集数据、资料分析和解释
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