摘要： 目的 构建小鼠Spata7基因视网膜内敲除模型，探讨Spata7基因突变导致光感受器细胞凋亡的机制。方法 以腺相关病毒（AAV）为载体搭载CRISPR/Cas9系统，注射1 μL AAV病毒混合液（3×10¹³ vg/mL）于出生14 d小鼠视网膜中，对Spata7基因进行视网膜内特异性敲除。1个月后行视网膜眼底照相观察AAV感染效率；定量PCR测定敲除效率；视觉电生理检测光感受器细胞功能；免疫荧光实验探究Spata7基因功能。结果 AAV有效搭载CRISPR/Cas9系统进入视网膜内并对Spata7基因进行敲除，敲除效率为54%。小鼠光感受器细胞大量死亡，导致整体光感受器细胞功能明显下降。敲除后，视紫红质无法被转运到光感受器细胞外节，引起明显的内质网应激。结论 Spata7基因敲除导致视紫红质蛋白运输到外节受阻而停留在内质网，并造成内质网应激，是导致光感受器细胞大量死亡的重要原因。

小鼠视网膜内Spata7基因敲除模型构建及其光感受器细胞凋亡机制的研究

刘和合, 张欣馨, 王游津, 孔珺

中国医科大学附属第四医院眼科, 沈阳 110005

收稿日期:2020-05-05出版日期:2020-12-30发布日期:2020-12-09
通讯作者:孔珺E-mail:kongjun@hotmail.com
作者简介:刘和合(1992-),女,硕士研究生.
基金资助:辽宁省光复眼病防治基金会横向课题（2900020001）


关键词: 视网膜色素变性, Spata7基因, 规律间隔成簇短回文重复序列, 内质网应激, 腺相关病毒
Abstract: Objective To construct a knockout model of Spata7 in vivo and explore the mechanism of apoptosis of photoreceptor cells induced by mutations of Spata7. Methods Adeno-associated virus (AAV) was used as a vector to deliver the clustered regularly interspaced short palindromic repeats (CRISPR) system into the retina to specifically knockout Spata7 in the retina. AAV mixture (1 μL) was injected into the P14 mouse retina. One month after injection,the efficiency of AAV infection,the function of photoreceptor cells,and the function of Spata7 were studied by retinal fundus imaging,electroretinogram (ERG),and immunofluorescence,respectively. Results AAV effectively carried the CRISPR system into the retina and knocked out Spata7 with a knockout efficiency of 54%. Numerous photoreceptor cells died in the retina,which led to a significant decline in the overall function of photoreceptor cells. Rhodopsin could not be transported to the outer segment of the photoreceptor cells,resulting in endoplasmic reticulum (ER) stress. Conclusion ER stress caused by the inability of rhodopsin to be transported to the outer segment and retained in the ER may be an important cause of photoreceptor cell death in a Spata7 retina-specific knockout model.
Key words: retinitis pigmentosa, Spata7 gene, clustered regularly interspaced short palindromic repeats, endoplasmic reticulum stress, adeno-associated virus
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