Slc4a11基因缺失在CHED发病中的作用

The role of Slc4a11 gene depletion in pathogenesis of congenital hereditary endothelial dystrophy

关键词:  Slc4a11基因  先天性遗传性角膜内皮营养不良  自噬

Key words:  Slc4a11 gene  congenital hereditary endothelial dystrophy  autophagy

摘要:

目的　探讨Slc4a11缺失在先天性遗传性角膜内皮营养不良（congenital hereditary endothelial dystrophy, CHED）发病中的作用及其与自噬信号通路的潜在调控关系。方法　利用CRISPR/Cas9技术构建Slc4a11基因敲除小鼠模型。以野生型小鼠（wild type, WT）和Slc4a11基因敲除小鼠（knockout, KO）为实验对象，通过Western印迹法、RT-qPCR、茜素红染色、H-E染色、透射电镜评估KO小鼠角膜表型特征的变化。通过Western印迹法和RT-qPCR检测自噬标志物LC3、beclin1、p62在角膜内皮的表达量。透射电镜观察两组小鼠角膜内皮细胞自噬情况。结果　Slc4a11基因敲除小鼠角膜显示与CHED相似的表型特征，包括角膜厚度增加；角膜内皮细胞体积增大，密度减小；后弹力层增厚；角膜内皮空泡形成。KO小鼠与WT小鼠相比，LC3、beclin1的mRNA和蛋白的相对表达量上升，差异有统计学意义（P<005），而p62的mRNA和蛋白的相对表达量下降，差异有统计学意义（P<005）。KO小鼠角膜内皮细胞内自噬体明显多于WT小鼠。结论　利用CRISPR/Cas9技术构建的Slc4a11基因敲除小鼠角膜显示CHED特征的表型。同时Slc4a11缺失通过增强自噬信号通路参与CHED的发病机制。

Abstract:

Objective　To investigate the role of Slc4a11 depletion in the pathogenesis of congenital hereditary endothelial dystrophy（CHED) and its relation with autophagy signaling pathway. Methods　The Slc4a11 gene-knockout mouse model was established with CRISPR/Cas9 gene targeting technique. Corneal abnormalities were evaluated using Western blotting, RT-qPCR, corneal endothelial cell staining, H-E staining and transmission electron microscopy. RT-qPCR and Western blotting were used to detect the expression of autophagy-related proteins LC3B, beclin1 and p62 in corneal endothelial cells. The autophagy of corneal endothelial cells was observed by transmission electron microscope. Results　Slc4a11 knockout mice showed similar clinical manifestations of human CHED, including corneal thickening, increasing endothelial cell area, decreasing endothelial cell density, thickening of DM and vacuolization of endothelial cells. Compared with wild type (WT) group, the mRNA and protein levels of LC3 and beclin1 were significantly higher in KO group（P<005), and the mRNA and protein levels of p62 were significantly lower in knockout (KO) group（P<005). TEM demonstrated that the autophagosomes in KO group were significantly more than those in WT group. Conclusion　In the study the Slc4a11 knockout mouse model has been constructed by CRISPR/Cas9 technique successfully, which presents the similar clinical manifestations of human CHED. The study also shows that Slc4a11 depletion is involved in the pathogenesis of CHED by enhancing autophagy signaling pathway.