VIP及受体VIPR2在小鼠正常屈光发育和形觉剥夺近视中的作用机制研究

Myopia is the leading causes of refractive error worldwide, and the pathogenesis is still unclear. Retina is very important in myopia, which can modulate growth and development of eye ball. In our preliminary experiments, we had found that expression level of Vasoactive Intestinal Peptide (VIP) was significant in RNA-Seq, the receptor of VIP (VIPR2) was associated with high myopia in Chinese population by genome-wide meta-study. Based on our preliminary study, we proposed our hypothesis that “VIP and VIPR2 in retina is involved in normal refractive error development and form deprived myopia”. Firstly, we clarify which kinds of cells can express VIP and VIPR2 protein in mice’s retina using immunofluorescence staining by two or three specific antibody which can bind with Marker protein which expressed in some kinds of retinal cells. Secondly, we will make clear whether VIPR2 can result to myopia by administrating agonist and antagonist of VIPR2 for mice. Then, in order to further confirm whether myopia caused by VIPR2, VIPR2 knockout mouse was used to study whether its refractive development was different from wildtype mice. Thirdly, in order to clarify the role of VIP and VIPR2 in mouse's retina, mouse for VIPR2 was only knockdown in retina was used. Furthermore, immunofluorescence staining for FDM mice's retina were carried out to ascertain which kinds of cell were associated with myopia, downstream signaling molecules of VIPR2 pathway were analyzed by quantitative Real Time PCR, Western Blot and immunofluorescence staining to clarify which pathway is involved in myopia, Finally, clarify the role of VIP and VIPR2 in myopia, further elucidate the pathogenesis of myopia, and offer the theoretical basis for drug intervention in myopia.

近视是常见的一种屈光不正，其致病机制仍不明确。视网膜调控眼球生长与发育，在近视形成中起重要作用。我们前期工作中发现在小鼠视网膜转录组数据中VIP表达存在明显差异，全基因组关联分析证实其受体VIPR2与近视相关。本项目在此基础上提出“视网膜VIP及受体VIPR2参与屈光发育调控及形觉剥夺性近视形成”的工作假说。将首先明确VIP及VIPR2在视网膜何种细胞上表达；给予小鼠VIPR2激动剂和拮抗剂推测VIPR2是否是近视形成的原因；通过全身基因敲除小鼠，进一步明确VIPR2是否参与近视形成；利用视网膜特异敲减VIPR2基因，明确视网膜VIPR2在近视中的作用；利用FDM小鼠，免疫荧光确定视网膜哪类细胞参与近视，WB等检测VIPR2信号通路分子，明确VIPR2通过哪条信号通路在近视中发挥作用，最终明确VIP及VIPR2在近视中的作用机制。进一步阐明近视的发病机制，为近视药物干预奠定理论基础。

近视是导致人眼屈光不正的最主要原因。它主要由基因和环境及其相互作用引起，目前对其发病机制知之甚少。使用Meta关联分析，我们发现中国汉族人群中VIPR2的SNP rs6979985与高度近视相关（random-effect model P = 0.013）。qRT-PCR发现C57BL / 6J小鼠的视网膜VIP mRNA在形觉剥夺（FD）1天或2天时表达下调。在FD-2天的小鼠中，视网膜单细胞转录组测序显示VIPR2主要在双极细胞表达，而某些表达VIPR2的细胞亚类的cAMP途径受到明显抑制。VIPR2拮抗剂PG99-465腹腔注射可引起C57BL / 6J小鼠产生相对性近视。相反，VIPR2激动剂Ro25-1553则可抑制FD近视的进展。与野生型同窝仔相比，VIPR2基因敲除（VIPR2-KO）小鼠的屈光度明显向漂移近视（平均漂移约-3.24屈光度）（P < 0.05）。对VIPR2-KO小鼠的视网膜RNA-Seq进行分析表明，视觉感知明显增强，并且光刺激和眼睛发育的感觉感知明显富集。在暗视条件下，在7周龄时VIPR2-KO小鼠的a波和b波振幅明显高于野生型同窝仔动物（P < 0.05）。因此，我们推测VIPR2的功能障碍会影响双极细胞的功能，导致异常的电生理影响视觉信号的处理和转导，这些变化最终导致了近视的发展。