利用Cre/loxP系统定位腺苷A2A受体在小鼠视网膜上的分布

目的：运用Cre/loxP系统来定位腺苷A2A 受体（A2AR）在小鼠视网膜上的分布情况。方法：实验研究。将雄性B6.FVB（Cg）-Tg（Adora2a-cre KG139Gsat/Mmucd（简称A2A-cre）小鼠与雌性B6．129S6-Gt（ROSA）26Sortm9（CAG-tdTomato）Hze/J（简称Ai9）小鼠交配，对新生小鼠基因型进行鉴定，并选取5 只4 周龄A2A-cre+，Ai9 鼠，此小鼠表达的Cre酶能敲除Ai9 小鼠中的STOP序列使Tomato荧光蛋白表达。采用免疫荧光方法分别将Tomato红色荧光蛋白与不同类型的视网膜细胞抗体共标记，通过观察Tomato红色荧光蛋白和视网膜细胞特异性抗体的共定位结果，来明确A2AR在视网膜上的定位情况。结果：免疫荧光结果显示Tomato+细胞主要分布在视网膜神经节细胞层和内核层，外核层并没有发现。其中神经节细胞层中的Tomato+细胞少数为神经节细胞，多数为异位无长突细胞，而分布于内核层的Tomato+细胞主要为Müller细胞和无长突细胞。进一步对无长突细胞亚型分析发现，Tomato+无长突细胞多数为AⅡ型无长突细胞，而胆碱能无长突细胞和多巴胺能无长突细胞并无Tomato+表达。结论：在4 周龄A2A-cre+，Ai9 小鼠视网膜中，A2AR主要表达于神经节细胞、无长突细胞和Müller细胞中。

Localizing the Distribution of the Adenosine A2A Receptor in the Mouse Retina Using a Cre/loxP System

Objective:To localize the distribution of the adenosine A2A receptor (A2AR) in the mouse retina using a Cre/loxP system.Methods:In this experimental study,male A2A-cre mice [B6.FVB (Cg)-Tg (Adora2a-cre) KG 139Gsat/Mmucd] were mated with female Ai9 mice [B6.129S6-Gt (ROSA) 26Sortm9 (CAG-tdTomato) Hze/J].The genotypes of the newborn mice were identified and five A2A-cre+,Ai9 mice were chosen at 4 weeks of age.The Cre enzyme of the mice can knock out the STOP sequence of Ai9 mice,which will cause the expression of the tomato fluorescent protein.The localization of A2AR in the retina can be determined through the double immtmostaining of the tomato red fluorescent protein and markers of different retinal cells.Results:Immunofluorescence microscopy showed that Tomato+ cells were mainly distributed in the ganglion cell layer and inner nuclear layer,and not in the outer nuclear layer.In the ganglion cell layer,the minority of Tomato+ cells were ganglion cells while the majority were ectopic amacrine cells.In the inner nuclear layer,Tomato+ cells were mainly Müller and amacrine cells.With further analysis of the amacrine cell subtypes,we discovered that most of the Tomato+ cells were AⅡ amacrine cells,not cholinergic or dopaminergic amacrine cells.Conclusions:This study suggests that A2AR is expressed in ganglion cells,amacrine cells,and Müller cells in 4-week-old mouse retinas.