本文選題：原發(fā)性肝內膽管結石　切入點：ABCB11　出處：《第三軍醫(yī)大學》2017年碩士論文

【摘要】：一、研究目的ABCB11基因編碼的蛋白質(bile salt export pump,BSEP)的功能主要是膽汁轉運,而其基因突變與膽汁淤積及膽石癥發(fā)病密切相關。前期研究中也表明ABCB11基因在原發(fā)性肝內膽管結石患者中存在基因突變,并且影響到編碼蛋白的表達。因此,本研究目的重點是深入探討ABCB11基因突變與原發(fā)性肝內膽管結石發(fā)病的聯(lián)系,以及研究ABCB11基因突變影響其編碼蛋白BSEP在細胞定位、表達的機制。二、研究方法1.收集我院收治的原發(fā)性肝內膽管結石患者443例(入院以后均經(jīng)術前影像學檢查明確診斷),同時收集我院健康體檢中心健康人群560例為對照組(均排除慢性肝病及膽石癥)。收集上述PIS患者和健康人群的外周血液5ml,立即分離淋巴細胞,放-80℃冰箱保存。收集統(tǒng)計原發(fā)性肝內膽管結石患者相關臨床資料。2.提取淋巴細胞DNA,并通過對ABCB11基因全外顯子測序,檢測病例組(原發(fā)性肝內膽管結石患者)與對照組(健康人群)ABCB11基因突變,在此基礎上進一步分析ABCB11基因突變與臨床資料間的關聯(lián)。3.對原發(fā)性肝內膽管結石患者的新鮮冰凍肝組織提取總m RNA與膜蛋白,并采用定量PCR技術與western blot技術檢測原發(fā)性肝內膽管結石患者ABCB11基因的m RNA與BSEP的表達,在此基礎上進一步分析與ABCB11基因突變的相關性。4.構建包含人的ABCB11基因全部開放閱讀框的野生型質粒,通過定點突變再構建突變型質粒。HEK293細胞分別轉染野生型和突變型質粒,轉染48小時后提取總m RNA與膜蛋白,并運用定量PCR技術與western blot技術檢測突變型與野生型質粒的mRNA與BSEP的表達。轉染MDCK細胞分別轉染野生型和突變型質粒,轉染48小時后進行免疫染色,采用激光共聚焦顯微鏡掃描技術觀察BSEP在MDCK細胞的分布。三、研究結果1.原發(fā)性肝內膽管結石患者與健康人群相比ABCB11基因存在兩個有意義的突變位點,分別是錯義突變rs118109635(p=0.025)與同義突變rs497692(p=0.006),并且這兩個突變位點與術前黃疸具有密切相關性(p=0.026,p=0.011)。2.發(fā)生錯義突變rs118109635的原發(fā)性肝內膽管結石患者肝組織BSEP表達降低,m RNA表達水平無變化,而發(fā)生同義突變rs497692的原發(fā)性肝內膽管結石患者肝組織m RNA與BSEP表達都降低。3.在細胞水平我們也發(fā)現(xiàn)突變rs118109635(A865V)使BSEP在HEK293細胞膜上表達降低,在MDCK細胞膜上分布減少,而m RNA表達水平無變化。四、研究結論ABCB11基因突變(rs118109635和rs497692)與原發(fā)性肝內膽管結石存在密切聯(lián)系,并且與術前黃疸具有密切相關性。同時,具有rs118109635或rs497692突變的原發(fā)性肝內膽管結石患者肝組織BSEP的表達均明顯降低。此外,體外實驗也表明突變rs118109635使BSEP在細胞膜上表達以及分布減少。因此,ABCB11基因的突變rs118109635、rs497692可能是PIS的發(fā)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，

本文編號：1722581