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

Mechanism of apoptosis in retinal photoreceptor cells in Spata7 knockout mice

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1μL 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。

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(1μL)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.