铜伴侣蛋白CCS基因多态性对剥脱综合征形成影响及其作用机制研究

Exfoliation syndrome (XFS), which is often complicated by cataract and zonular defect, leads to a high blindness rate and poor surgical outcomes. Although LOXL1 gene has been recognized as a susceptible gene, our previous research has found that LOXL1 gene is normal in part of the XFS patients. However, it was abnormal for the copper chaperone for superoxide dismutase（CCS） gene, who is the provider of the copper prosthetic group and the SOD enzyme copper chaperone as well. As a basis of preliminary studies, we propose the hypothesis that the abnormality of CCS gene, leading to a disorder of copper ion transportation, can directly or indirectly influence the function of LOXL1 protein. Our plan is listed as follow. Firstly, venous blood of XFS patients and control group is obtained to extract DNA in order to detect LOXL1 and CCS gene polymorphism. Secondly, we conduct observation of human lens epithelial cells (HLEC) about the intracellular SOD activity and the methylated and expressive changes of LOXL1 gene after CCS gene knockout. Furthermore, lenti-guide-CCS-sgRNA virus is injected into the anterior chamber of CRISPR/Cas9 mice to observe the morphologic and functional changes of the lens and zonule after CCS gene knockout. Finally, reexpression in HLECs and the anterior chamber of mice after CCS gene knockout is detected to verify the function of CCS gene. Our aim is to elucidate the effect and pathogenesis of CCS gene for the XFS patients and provide the basic theory for early nonsurgical intervention.

剥脱综合征（XFS）常并发白内障伴悬韧带病变，致盲率高，手术疗效差。LOXL1基因是公认的易感基因，本项目组前期研究发现部分XFS患者LOXL1基因正常，但其辅基铜离子提供者铜伴侣蛋白CCS基因异常。CCS也为SOD酶铜分子伴侣。据此提出“铜伴侣蛋白CCS基因异常，铜离子转运异常，直接和间接影响LOXL1功能，发生XFS”假说。拟首先提取XFS与对照组患者静脉血DNA行LOXL1、CCS基因多态性检测；其次观察人晶状体上皮细胞（HLEC）CCS基因敲除细胞内SOD活性、LOXL1基因甲基化及其表达等改变；再次在CRISPR/Cas9小鼠前房内注入慢病毒CCS-sgRNA，观察CCS基因敲除后晶状体、悬韧带形态及功能改变。最后HLEC及小鼠前房CCS基因敲除后再表达做功能验证。旨在阐明CCS基因在XFS发病过程中的作用及机制，为早期非手术干预提供依据。

剥脱综合征(XFS)常并发白内障伴悬韧带病变，致盲率高，手术疗效差。项目组前期研究发现其辅基铜离子提供者铜伴侣蛋白CCS基因异常，据此提出铜伴侣蛋白CCS基因异常，铜离子转运异常，直接和间接影响LOXL1功能，发生XFS。本课题组将CCS基因异常与LOXL1功能相结合，突破传统XFS研究的局限性，为XFS发病机制提供新思路。. 本研究主要内容包括：（1）提取XFS与对照组患者静脉血及晶状体前囊膜行CCS蛋白水平检测;（2）建立人晶状体上皮细胞(HLEC) CCS基因敲除细胞，并检测SOD活性、LOXL1基因甲基化及其表达等改变;（3）建立CCS基因敲除小鼠，观察CCS基因敲除后晶状体、悬韧带形态及功能改变。. 项目组首次阐明CCS基因通过对LOXL1基因启动子以及第一外显子区甲基化的调控，针对LOXL1表达水平施加影响，使LOXL1 mRNA和蛋白表达水平降低；同时，项目组明确了CCS基因异常使SOD1降低，引起眼内氧化-抗氧化失衡，诱导氧化应激，并通过Fas介导的途径激活Caspase-3，促进HLEC凋亡。项目组证实了CCS基因通过直接促进HLEC凋亡、间接影响LOXL1功能两条途径，阐明了CCS在XFS发病过程中的作用。本项目为XFS发病机制提供新思路，有助于解决XFS患者发病机制这一难题，为早期非手术干预提供依据，减轻社会医疗负担，具有重要社会意义。