CYP1B1基因缺失小鼠在高糖皮质激素环境下对青光眼的易感性

Susceptibility of CYP1B1 knock-out mouse to glaucoma in a high glucocorticoid environment

目的观察高浓度糖皮质激素对CYP1B1-/-小鼠青光眼易感性的影响。方法采用成年CYP1B1-/-小鼠作为实验模型,以同龄C57BL/6J小鼠作为对照。每3天结膜下注射0.04mL倍他米松,用Tonopen眼压(IOP)笔每周定期测定小鼠IOP,于第一次给药前,给药后4、8、12周分别摘除眼球,制备石蜡切片,光学显微镜下观察小鼠视网膜形态和厚度,采用TUNEL法检测视网膜神经节细胞(RGCs)的凋亡。结果与给药前相比,2组小鼠给药后4、8、12周的IOP均较前升高,差异有统计学意义(P<0.05);CYP1B1-/-小鼠的IOP在给药后8周、12周较C57BL/6J小鼠显著升高,差异有统计学意义(P<0.05)。随着倍他米松注射时间的推移,2组小鼠视网膜纤维层均变薄,各时间组的总体差异有统计学意义(P<0.05);且CYP1B1-/-小鼠视网膜纤维层厚度在给药后8周、12周较C57BL/6J小鼠显著变薄,差异有统计学意义(P<0.05)。2组小鼠RGCs凋亡的速度与给药前相比均显著增加,差异有统计学意义(P<0.05);且CYP1B1-/-小鼠与C57BL/6J小鼠相比结果更为显著(P<0.05)。结论在高浓度糖皮质激素的诱导下,CYP1B1-/-小鼠对青光眼的易感性增加。

Background Genetic factors involves the pathogenesis of primary congenital glaucoma（PCG）. It has already been confirmed that CYP1B1 is located in GLC3A, one of the disease-causing genes on PCG-related loci. However, whether the occurrence of PCG is caused directly by the CYP1B1 mutations or with other genetic or environmental factor remains unclear. Objective The present study was to investigate the susceptibility of glaucoma in CYP1B1 gene knock-out mouse under high dosage of glucocorticoid environment. Methods Glaucoma models were induced by subconjunctival injection of 0.04 mL betamethasone for 4 times at 3-day interval in the right eyes of 40 SPF adult CYP1B1^-/- mice. Forty SPF adult C57BL/6J mice with the same treatment method were as control. The intraocular pressure （IOP） of mice were measured by Tono-pen XL applanometer once a week before and after the injection. The animals were sacrificed, their eyeballs were enucleated, and retinal thickness were evaluated under the light microscope before and 4 weeks, 8 weeks, 12 weeks after injection respectively. The apoptosis of retinal ganglion cells（RGCs） was detected by terminal deoxnucleotidyl transferase mediated dUTP biotin nick end labeling（ TUNEL）. This experiment followed Guide to the Care and Use of Experimental Animals（ NIH publication No. 23 -85, revised 1996）. Results The IOP was persistently elevated in 4,8 and 12 weeks after the injection of betamethasone in comparison with before injection in both CYP1B1^-/- mice and C57BL/6J mice （P 〈 0.05） , and the IOP of CYP1B1^-/- mice was significantly higher than that of C57BL/6J mice in 8 and 12 weeks after the injection of betamethasone（ P 〈 0.05 ）. Retinal fibrous layer was gradually thinner with the prolong of time after injection of betamethasone in both CYP1B1^-/- mice and C57BL/6J mice （P 〈 0.05 ）. The thickness of retinal fibrous layer in CYP1B1^-/- mice was obviously thinner than that of C57BL/6J mice in 8 and 12 weeks after the injection of betamethasone （ P 〈 0. 05 ）. Apoptosis rate of RGCs was increased after the injection of betamethasone compared with before the injection in both CYP1B1^-/- mice and C57BL/6J mice （ P 〈 0.05 ） , especially in CYPIB1^-/-mice（ P 〈 0.05 ）. Conclusion In an environment of high level glucocorticoid, the susceptibility to glaucoma is increased higher in CYP1B1^-/- mice.