C57BL/6小鼠形觉剥夺性近视视网膜Pax6的表达及其启动子区域CpG岛甲基化的改变

目的研究C57BL/6小鼠形觉剥夺性近视形成和恢复期视网膜Pax6 mRNA表达及其启动子区域CpG岛甲基化水平的变化情况。方法实验研究。采用单眼形觉剥夺（MD）建立C57BL/6小鼠近视动物模型和恢复期动物模型，将入选的小鼠按随机数字表法分为：MD 4周实验组（12只）、同龄对照组（12只）；MD 4周再恢复1周实验组（6只）、同龄对照组（NC，6只）。实验前后分别用红外偏心摄影验光仪检测小鼠眼球的屈光状态，光学相干断层扫描（OCT）检测小鼠的眼轴长度和玻璃体腔深度。RT-PCR检测视网膜 Pax6 mRNA表达水平；用硫化测序聚合酶链式反应（BSP）检测C57BL/6小鼠视网膜Pax6启动子区CpG岛甲基化水平。实验组形觉剥夺眼（MD-T）与对侧眼（MD-C）比较采用配对t检验，不同组小鼠比较采用独立样本t检验。结果MD 4周后，MD-T屈光度（-3.27±0.52）D]与MD-C（1.13±1.17）D]（t=-12.726，P<0.01）、NC（1.65±1.69）D]（t=-6.832，P<0.01）相比明显向近视方向漂移。MD 4周再恢复1周后，实验性近视完全恢复。MD 4周后，MD-T相对定量值（0.495±0.247）相比MD-C（1.011±0.477）（t=-3.16，P<0.05）、NC（1.071±0.401）（t=-2.99，P<0.05）Pax6 mRNA表达水平明显下降；而恢复1周后Pax6 mRNA表达水平恢复到正常对照水平。MD 4周后，MD-T Pax6启动子区CpG岛甲基化水平与MD-C、NC比较，差异无统计学意义。结论形觉剥夺性近视C57BL/6小鼠视网膜Pax6 mRNA表达明显下降，启动子区CpG岛的甲基化水平无明显变化，提示近视形成可能和Pax6的下降相关，但这一改变可能不是由视网膜Pax6启动子区CpG岛的甲基化变化引起。

Study on mRNA expression and DNA methylation of Pax6 in form-deprived myopia in C57BL/6 mice

ObjectiveTo investigate the changes in refraction and ocular biometric parameters in monocular form-deprivation myopia， and to investigate whether myopia development is associated with changes in the retinal levels of Pax6 messenger RNA （mRNA） and the DNA methylation state in cytosine-phosphate-guanine （CpG） sites in the Pax6 promoter. MethodsExperimental study. A diffuser lens was worn over one eye of one group of C57BL/6 mice for 4 weeks to establish a monocular form deprivation （MD） myopia model， and another group wore a diffuser lens for 4 weeks， then the goggles were removed for 1 week to build a recovery model. A total of 36 C57BL/6 mice （postnatal day 21） were randomly assigned to the MD group （n=12）， a normal control （NC） group （n=12）； and MD recovery group （n=6）， a corresponding control group （n=6）. Ocular biometry was determined in mice by an EIR （eccentric infrared photorefractor） and OCT （optical coherence tomography）. The Pax6 mRNA level in the retina was determined by real-time PCR， and the Pax6 methylation state was observed by bisulfite DNA sequencing. Data were analyzed using paired t test or independent t test. ResultsAfter 4 weeks of form deprivation， a significant myopic shift had been induced in the treated eyes （MD-T） （-3.27±0.52 D） compared to the contralateral eyes （MD-C） （1.13±1.17 D） （t=-12.726， P<0.01） and normal control eyes （NC） （1.65±1.69 D） （t=-6.832， P<0.01）. Pax6 mRNA in the MD-T eyes （0.495±0.247） decreased significantly compared to the MD-C eyes （1.011±0.477） （t=-3.16， P<0.05） and the NC eyes （1.071±0.401） （t=-2.99， P＜0.05）. One week after the removal of the diffuser lenses， Pax6 mRNA expression in recovery eyes had been fully restored to levels in the control eyes. The methylation percentage in CpG sites of the Pax6 promoter in the MD-T eyes was not significantly different compared to MD-C and NC eyes. ConclusionMonocular form deprivation myopia can be induced in C57BL/6 mice. Retinal Pax6 mRNA was reduced in the MD-T eyes compared to MD-C and NC eyes， but the changes in Pax6 mRNA may not be caused by DNA methylation of the CpG island in the promoter region of Pax6.