Vipr2敲除对小鼠视网膜功能的影响

目的：检测血管活性肠肽受体2（Vipr2）敲除小鼠多巴胺（DA）等神经递质含量和视网膜电生理，明 确Vipr2敲除对视网膜功能的影响。方法：实验研究。利用RT-PCR检测血管活性肠肽（Vip）、Vipr2 在4周龄雄性C57BL/6J小鼠眼球组织中的表达水平。通过CRISPR-Cas9基因编辑技术构建Vipr2敲 除（Vipr2-KO）小鼠，然后在4周龄时检测Vipr2-KO和Vipr2野生型（Vipr2-WT）小鼠视网膜中DA等 神经递质含量及视网膜电生理功能。Vipr2-KO小鼠与Vipr2-WT小鼠DA等神经递质含量比较采用独 立样本t检验，而2种小鼠视网膜电生理数据比较采用双因素重复测量方差分析。结果：Vip和Vipr2 mRNA在小鼠视网膜、脉络膜+视网膜色素细胞、角膜和巩膜中均有表达；在虹膜和晶状体中Vipr2 呈低表达，未检测到Vip表达。与Vipr2-WT小鼠相比，Vipr2-KO小鼠表现为近视（t=2.51，P=0.017）， 视网膜DA、3, 4-二羟基苯乙酸（DOPAC）、高香草酸（HVA）含量均明显升高（t=3.42，P=0.001； t=2.15，P=0.037；t=3.27，P=0.002），DOPAC/DA比值无变化；而玻璃体液中DA、DOPAC、DOPAC/ DA、HVA、去甲肾上腺素含量差异均无统计学意义。视网膜和玻璃体液中天冬氨酸、谷氨酸、丝氨 酸、谷氨酰胺、甘氨酸、牛磺酸和γ氨基丁酸含量在Vipr2-KO小鼠与Vipr2-WT小鼠之间差异均无统 计学意义。在暗适应不同光刺激强度下（-3.699、-2.201、-0.699、0.301、0.799 log cd·s/m2 ），Vipr2- KO小鼠相比Vipr2-WT小鼠，b波振幅明显增加（F=8.65，P=0.015）。结论：Vipr2敲除可引起4周龄 小鼠屈光向近视发展，影响视网膜中DA、DOPAC、HVA合成代谢，并引起视网膜电生理功能异常。 提示Vipr2敲除可能通过影响视网膜功能参与近视形成，但具体作用机制有待进一步研究。

The Effect of Vipr2 Knockout on Retinal Function in Mice

Objective: To determine if the loss of function of retinal vasoactive intestinal peptide receptor 2 (Vipr2) affects dopamine (DA) turnover and retinal electrophysiology behavior. Methods: This is an experimental study. RT-PCR analyzed vasoactive intestinal peptide (Vip) and Vipr2 mRNA expression levels in different ocular tissues of 4- week-old male wildtype C57BL/6J mice. CRISPR-Cas9 gene-editing technology constructed the Vipr2-KO mice. Their refraction, turnover of retinal dopamine, and electroretinograms were measured and compared in these two different groups of 4-week-old Vipr2-KO and Vipr2-WT mice. Statistical analysis of the refraction and DA turnover used the independent sample t-test, and the data of retinal electrophysiology were evaluated with the two-way repeated measures ANOVA between these twodifferent groups of mice. Results: Vip and Vipr2 mRNA levels were highly expressed in the retina, choroid/ retinal pigment cells, cornea, and sclera. In the iris and lens, Vipr2 mRNA was expressed at very low levels whereas the Vip mRNA expression level was undetectable. The refraction of 4-week-old Vipr2-KO mice was significantly shifted more towards myopia than that in the Vipr2-WT mice (t=2.51, P=0.017). The contents of DA, 3, 4-dihydroxyphenylacetate (DOPAC), and homovanillic acid (HVA) were significantly increased more in the 4-week-old Vipr2-KO mice than in the Vipr2-WT mice (t=3.42, P=0.001; t=2.15, P=0.037; t=3.27, P=0.002, respectively). However, the levels of DA, DOPAC, DOPAC/DA, HVA, and norepinephrine in the vitreous humor were not altered between these two different groups. The amino acid contents of aspartic acid, glutamic acid, serine, glutamine, glycine, taurine, and γ-aminobutyric acid were invariant in the retina and vitreous humor of these groups. The b-wave amplitudes under scotopic conditions (-3.699, -2.201, -0.699, 0.301, 0.799 log cd·s/m2 ) were higher in the 4-week-old Vipr2- KO mice than in its Vipr2-WT counterpart (F=8.65, P=0.015). Conclusions: Loss of Vipr2 function induces a refractive shift towards myopia at age 4 weeks, and alters both the retinal DA turnover and electroretinogram pattern. These changes suggest that loss of Vipr2 function alters retinal function and is thereby involved in myopia progression, but its mode of action requires further study.