ABCB11基因突变致PFIC2患儿胆盐输出泵蛋白膜定位异常的机制研究

Progressive familial intrahepatic cholestasis (PFIC) accounts for 10 to 15% of children cholestasis pathogens, is an autosomal-recessive type of intrahepatic cholestasis of hepatocellular origin, and PFIC represents 10- 15% of liver transplantation indications in children. In cases of PFIC type 2 (PFIC2), the initial presentation and its evolution appear to be more severe, with permanent jaundice from the first months of life and the rapid appearance of liver failure, also the early hepatocellular carcinoma (before 1 year of age) and cholangiocarcinoma may complicate the course of PFIC2. The pathogenesis of PFIC2 is not only a hot topic, but also a difficult one.. PFIC2 is caused by mutations in the ABCB11 gene. The ABCB11 gene encodes the ATP- dependent canalicular BSEP in human liver and is located on human chromosome 2. BSEP is exclusively expressed in hepatocytes and is mainly localized at the canalicular membrane, reduced or even absent expression of canalicular BSEP is a typical feature of PFIC2. Mutations of BSEP are responsible for the decreased biliary bile salt secretion described in affected patients, leading to decreased bile flow and accumulation of bile salts within the hepatocyte with ongoing severe hepatocellular damage.. The locationization of BSEP in the canalicular membrane is regulated by HCLS1-associated protein X-1 (HAX-1). Depletion of HAX-1 leads to an increase of BSEP at the apical membrane in MDCK cells. Further study revealed when C57L mice were fed with a high cholesterol, BSEP and HAX-1 showed enhanced interaction in the hepatocytes, which than affected the endocytosis and localization of BSEP from the canalicular membrane...In prophase research, we found a PFIC2 boy with two novel ABCB11 mutations, c.1415A>G（Y472C）及c.3392A>T（D1131V）, with direct sequencing; The liver biopsy revealed BSEP expression decreased significantly in the hepatocyte membranes. Protein structure predicted showed that Y472C and D1131V mutations can lead to structure change of BSEP linker domain, which is HAX-1 binding region in BSEP, suggest BSEP mutations may affect HAX - 1 protein affinity and than influence its membrane localization.. Surface plasmon resonance (SPR) is a unique, optoelectronic, label-free, noninvasive, direct-reading enrichment detection method that utilizes interaction between proteins, combined with immunoprecipitation , it will be a good tools to analyze the interaction between HAX-1 and mutated BSEP. In this study, BSEP Y472C and D1131V mutant cells model and transgenic mice model were built to detect the protein affinity between BSEP and HAX-1 and its affect on the localization of mutation BSEP. SPR, immunoprecipitation and confocal laser scanning technology will be preformed to inspect the relationship between the BSEP and HAX-1 the affinity and BSEP canalicular localization. It will cast a new light on the pathogenesis of PFIC2 and finally benefit the therapy and outcome of PFIC2 patients.

进行性家族性肝内胆汁淤积症2型（PFIC2）是由ABCB11基因编码的胆盐输出泵蛋白（BSEP）突变引起。在前期工作中课题组对PFIC2患儿肝活检显示BSEP在肝细胞膜上表达明显减少, BSEP的膜定位异常，基因测序发现BSEP新突变Y472C及D1131V。蛋白三维结构分析表明，Y472C及D1131V突变皆可导致BSEP胞内连接域的结构改变，HS1 相关蛋白X－1（HAX-1）可以特异结合于BSEP胞内连接域，两者亲和力改变对BSEP的膜定位发挥关键作用。提示BSEP突变有可能影响HAX-1蛋白亲和力而导致其膜定位异常。本研究通过构建BSEP蛋白Y472C及D1131V突变型的细胞模型及转基因小鼠模型；综合运用表面等离子共振、激光共聚焦及免疫共沉淀等技术，观察不同突变型BSEP和HAX-1蛋白的亲和力变化及其对BSEP膜定位的影响，旨在探讨BSEP突变在PFIC2患者中的致病机理。

胆盐输出泵蛋白(BSEP)是肝细胞膜上主要的胆盐外运载体，目前已知进行性家族性肝内胆汁淤积症2型(PFIC2)是由编码BSEP的ABCB11基因突变所致。PFIC2患儿出生即可发生进行性加重性黄疸，可很快进展为肝衰竭，目前尚无有效的治疗方法。BSEP几乎均特异性的表达于肝细胞毛细胆管面上，主要功能是与单价胆汁酸盐结合，通过水解 ATP 将胆盐逆浓度梯度泵入毛细胆管内。.首先，为了明确ABCB11基因c.1415A>G(p.Y472C)、c.3392A>T(p.D1131V)和c.2005A>G(p.I669V)变异的致病机制。我们在细胞水平明确了这些变异对BSEP蛋白表达及定位的影响。其次，为明确 HAX-1 蛋白和野生型及不同突变型 BSEP 蛋白体外相互作用，我们运用siRNA沉默HAX-1以及COIP技术在体外检测其相互作用。最后，我们通过在体外给予野生型及突变型最适浓度及最适时间的药物刺激，从而探讨了4-PBA和VX-809对不同BSEP突变型的纠正作用。