ABCB11基因突变与原发性肝内胆管结石发病机制的相关性研究

发布时间：2018-04-08 17:20

  本文选题：原发性肝内胆管结石　切入点：ABCB11　出处：《第三军医大学》2017年硕士论文

【摘要】：一、研究目的ABCB11基因编码的蛋白质(bile salt export pump,BSEP)的功能主要是胆汁转运,而其基因突变与胆汁淤积及胆石症发病密切相关。前期研究中也表明ABCB11基因在原发性肝内胆管结石患者中存在基因突变,并且影响到编码蛋白的表达。因此,本研究目的重点是深入探讨ABCB11基因突变与原发性肝内胆管结石发病的联系,以及研究ABCB11基因突变影响其编码蛋白BSEP在细胞定位、表达的机制。二、研究方法1.收集我院收治的原发性肝内胆管结石患者443例(入院以后均经术前影像学检查明确诊断),同时收集我院健康体检中心健康人群560例为对照组(均排除慢性肝病及胆石症)。收集上述PIS患者和健康人群的外周血液5ml,立即分离淋巴细胞,放-80℃冰箱保存。收集统计原发性肝内胆管结石患者相关临床资料。2.提取淋巴细胞DNA,并通过对ABCB11基因全外显子测序,检测病例组(原发性肝内胆管结石患者)与对照组(健康人群)ABCB11基因突变,在此基础上进一步分析ABCB11基因突变与临床资料间的关联。3.对原发性肝内胆管结石患者的新鲜冰冻肝组织提取总m RNA与膜蛋白,并采用定量PCR技术与western blot技术检测原发性肝内胆管结石患者ABCB11基因的m RNA与BSEP的表达,在此基础上进一步分析与ABCB11基因突变的相关性。4.构建包含人的ABCB11基因全部开放阅读框的野生型质粒,通过定点突变再构建突变型质粒。HEK293细胞分别转染野生型和突变型质粒,转染48小时后提取总m RNA与膜蛋白,并运用定量PCR技术与western blot技术检测突变型与野生型质粒的mRNA与BSEP的表达。转染MDCK细胞分别转染野生型和突变型质粒,转染48小时后进行免疫染色,采用激光共聚焦显微镜扫描技术观察BSEP在MDCK细胞的分布。三、研究结果1.原发性肝内胆管结石患者与健康人群相比ABCB11基因存在两个有意义的突变位点,分别是错义突变rs118109635(p=0.025)与同义突变rs497692(p=0.006),并且这两个突变位点与术前黄疸具有密切相关性(p=0.026,p=0.011)。2.发生错义突变rs118109635的原发性肝内胆管结石患者肝组织BSEP表达降低,m RNA表达水平无变化,而发生同义突变rs497692的原发性肝内胆管结石患者肝组织m RNA与BSEP表达都降低。3.在细胞水平我们也发现突变rs118109635(A865V)使BSEP在HEK293细胞膜上表达降低,在MDCK细胞膜上分布减少,而m RNA表达水平无变化。四、研究结论ABCB11基因突变(rs118109635和rs497692)与原发性肝内胆管结石存在密切联系,并且与术前黄疸具有密切相关性。同时,具有rs118109635或rs497692突变的原发性肝内胆管结石患者肝组织BSEP的表达均明显降低。此外,体外实验也表明突变rs118109635使BSEP在细胞膜上表达以及分布减少。因此,ABCB11基因的突变rs118109635、rs497692可能是PIS的发病因素。
[Abstract]:Objective: to study the function of salt export pumpP (BSEP) encoded by ABCB11 gene is mainly bile transport, and its gene mutation is closely related to cholestasis and cholelithiasis.Previous studies also showed that ABCB11 gene had gene mutation in patients with primary intrahepatic cholelithiasis and affected the expression of encoded protein.Therefore, the aim of this study was to explore the relationship between ABCB11 gene mutation and the pathogenesis of primary intrahepatic cholelithiasis, and to study the mechanism of ABCB11 gene mutation affecting the localization and expression of BSEP.Second, research method 1.A total of 443 patients with primary intrahepatic cholelithiasis in our hospital were collected. After admission, all the patients were diagnosed by preoperative imaging examination, and 560 healthy people in our health examination center were collected as control group (all excluding chronic liver disease and cholelithiasis).The peripheral blood fluid of above mentioned PIS patients and healthy people were collected 5 ml, lymphocytes were separated immediately and stored in-80 鈩，

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