TJP2基因突变在进行性家族性肝内胆汁淤积症中的致病机制研究

Progressive Familial Intrahepatic Cholestasis (PFIC) is an autosomal recessive disease that causes death and disability by childhood hepatopathy. Mutation of the gene TJP2 can lead to PFIC, but its mechanism has not been elucidated. In our previous research, 6 types of TJP2 mutations were discovered, two of the six mutations were found to be novel. All mutations were confirmed as pathogenic mutations by bioinformatics analysis. TJP2 interacts with many cellular structural proteins. Disruption of structural proteins and downstream pathways can lead to hepatic cell disfunction. Thus, we propose that mutation of TJP2 is central to PFIC. In this project, the mutational cell lines of HepG2 and THLE-3 will be constructed with the technology CRISPR/Cas9. The change of cell structure and Young's modulus caused by the mutation of TJP2 will be analyzed by the atomic force microscope. Morphological changes in the structure of mutant cells will be observed by the scanning electron microscope. The distribution of microfilaments and microtubules in those mutant cells will be analyzed by immunofluorescence. In all, uncovering the etiology of PFIC will provide better ways to diagnose and prevent childhood liver disease.

常染色体隐性遗传病进行性家族性肝内胆汁淤积症（PFIC）是儿童期肝病致死或致残的重要原因之一；TJP2为PFIC新致病基因，但是其具体作用机制尚未阐明。我们前期研究发现6种TJP2基因突变，包括2种新突变，经生物信息学分析鉴定都为致病性突变。紧密连接蛋白TJP2作为支架蛋白与许多细胞结构蛋白相连，并参与构建肝细胞结构。因此，我们推测TJP2基因突变引起肝细胞结构的异常在PFIC发病过程中发挥了关键作用。本项目利用CRISPR/Cas9方法构建突变型HepG2和THLE-3细胞株，采用原子力显微镜技术研究TJP2突变引起细胞形貌和杨氏模量的变化情况，再利用扫描电子显微镜技术研究TJP2基因突变导致细胞细微结构的异常情况，最后利用细胞免疫荧光技术研究TJP2基因突变改变细胞微丝和微管的分布情况。本研究结果将有助于进一步阐明PFIC的发病机制，为疾病诊断和预防提供科学依据。

TJP2基因突变导致进行性家族性肝内胆汁淤积症和妊娠期胆汁淤积症的发生，其具体的致病机制未明。1.本研究完成280例胆汁淤积症患儿和16例妊娠期胆汁淤积症孕妇的基因诊断，发现6例TJP2基因突变患儿，1例TJP2杂合变异孕妇，其中发现国际首次报道TJP2新突变4种。2.通过功能实验证明新突变c.1202A>G和c.2688-11A>G为致病性突变。利用crispr cas9技术成功构建新突变c.1202A>G细胞株，实验结果表明突变c.1202A>G导致TJP2 mRNA和蛋白均上调，细胞增殖能力增强，TJP2蛋白聚集细胞核内。利用Mini gene实验研究c.2688-11A>G新突变，实验结果发现该突变将内含子GATTCCTCTA剪切进mRNA,导致TJP2蛋白异常和截短。3.利用siRNA干扰技术下调TJP2蛋白后，P53蛋白表达量上调，Actin蛋白表达量不变，细胞内F-actin分散，细胞微丝改变，微丝排列紊乱，极性消失，细胞融合，细胞凋亡增加，细胞增殖能力减弱，细胞微管变化差异不大。免疫沉淀联合质谱实验发现TJP2和P53相互作用。原子力显微镜和扫描电子显微镜实验观察到细胞结构发生了改变。本项目的完成初步阐明了TJP2突变导致胆汁淤积症的发病机制，为治疗TJP2突变导致的胆汁淤积症提供了理论基础。