基因敲除技术研究ezrin维持晶状体结构的机制

As the next-generation sequencing technology, exome sequencing has been valued and applied widely in the field of genetics, including the genetic eye disorders. It has been successfully used in our last research. A mutant gene EZR was found in a four generation Chinese congenital cataract family. This is the first time that the EZR gene has been reported as a suspicious congenital cataract pathogenic cause in the world. However, this result still needs knockout technology to confirm further. Therefore, in this study, we will perform light microscopy, scanning electron microscopy, freeze-fracture transmission electron microscopy immunohistochemistry and molecular biological technology on the EZR knockout mouse to understand the protein ezrin well in the lens. The protein ezrin, one of the ERM proteins (ezrin, radixin and moesin) encoded by gene EZR,mainly presents in the young elongating cortical fiber cells and localizes to the plasma membranes of the lens. In recent years, the studies focused on the distribution and functions of ezrin in the lens reveals that ERM protein family especially ezrin plays a crucial role in the development of the lens, and its reduction or disfuction would potentially cause the disorder of the fiber cells which inducing the opacification of the lens. However, the exactly mechanism of ezrin in organizing lens cells and maintaining lens transparency is still unclear. We still don't know whether the EZR is a real cangenital catract pathogenic gene or not. Therefore, this study is important and necessary.

本课题申请人通过外显子组测序发现了基因EZR的突变可能导致一个四代中国人先天性核性白内障的发生，这是世界上对该基因是先天性白内障可疑致病基因的首次报道，但目前仍需通过EZR基因敲除鼠进一步验证该基因与先天性白内障的关系。EZR基因所编译膜细胞骨架连接蛋白ezrin是连接细胞膜和细胞骨架之间ERM蛋白家族成员之一。它与跨膜蛋白和细胞骨架蛋白的相互连接对膜结构域的形成有着极其重要的作用，因此ezrin作为连接蛋白可能对维持晶状体纤维细胞有序结构，保持晶状体的透明性有着十分重要的意义，但目前对其在维持晶状体透明性的具体机制尚不清楚。本课题拟利用条件性基因敲除技术，建立晶状体EZR基因敲除小鼠模型，从而利用光学显微镜、扫描电镜、冷冻蚀刻电镜、免疫组化及分子生物学方式观察基因敲除后晶状体纤维的有序组织结构的变化和晶状体透明性的改变，从而证实该基因是先天性白内障的致病基因。

本课题申请人通过外显子组测序发现了基因EZR的突变可能导致一个四代中国人先天性核性白内障的发生，这是世界上对该基因是先天性白内障可疑致病基因的首次报道，但目前仍需通过EZR基因敲除斑马鱼进一步验证该基因与先天性白内障的关系。EZR基因所编译膜细胞骨架连接蛋白ezrin是连接细胞膜和细胞骨架之间ERM蛋白家族成员之一。它与跨膜蛋白和细胞骨架蛋白的相互连接对膜结构域的形成有着极其重要的作用，因此ezrin作为连接蛋白可能对维持晶状体纤维细胞有序结构，保持晶状体的透明性有着十分重要的意义，但目前对其在维持晶状体透明性的具体机制尚不清楚。本项目利用基因敲除技术，建立晶状体EZR基因敲除斑马鱼模型，从而利用光学显微镜、投射电镜、免疫组化及分子生物学方式观察基因敲除后晶状体纤维的有序组织结构的变化和晶状体透明性的改变，从而证实该基因是先天性白内障的致病基因。.. 我们利用TALEN技术对斑马鱼的的两个等位基因：ezra和ezrb的移框突变导致其功能的丧失，从而达到敲除的效果。组织学显示突变的晶状体有核细胞数明显增加。突变的晶状体还显示异常的晶状体上皮细胞形态缺乏一个明确的过渡区，这表明在二次纤维分化的中断。突变的晶状体表现出增加的细胞增殖，表明突变晶状体内晶状体混浊与异常增殖以及分化细胞积累。这些结果表明，EZR在维持晶状体的正常结构起到了决定性的作用。