肝门部胆管癌经皮经肝胆道引流与内镜下逆行胆管引流胆汁中肿瘤细胞成瘤性对比与分析  

Comparison and Analysis of Tumorigenicity of Tumor Cells in Bile between PTBD and ERBD of Hilar Cholangiocarcinoma

作　　者：祝凯华 张德祥 陶颖 张舒龙 范坤 刘厚宝 ZHU Kai-Hua;ZHANG De-Xiang;TAO Ying;ZHANG Shu-Long;FAN Kun;LIU Hou-Bao(Department of General Surgery,Xuhui District Central Hospital,Shanghai 200031;Department of Biliary Surgery,Zhongshan Hospital,Fudan University,Shanghai 200032)

机构地区：[1]上海市徐汇区中心医院,普外科,上海200031 [2]复旦大学附属中山医院,胆道外科,上海200032

出　　处：《中国生物化学与分子生物学报》2025年第1期112-124,共13页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.82072682,82372832);上海市徐汇区卫生健康委员会项目(No.SHXH202005)资助。

摘　　要：肝门部胆管癌发病隐匿,常因胆汁淤积引起梗阻性黄疸,导致肝功能受损。对肿瘤合并恶性梗阻性黄疸,临床通常在手术前行胆汁引流。目前,胆汁引流方式主要是经皮经肝胆道引流(percutaneous transhepatic biliary drainage,PTBD)和内镜下逆行胆管引流(endoscopic retrograde biliary drainage,ERBD),2种引流方式各有优劣。PTBD引流易引起肿瘤种植转移,但机制不清楚。本文分析肝门部胆管癌PTBD及ERBD引流胆汁,从中分离获得肿瘤细胞,通过体外肿瘤球形成及体内移植瘤模型对PTBD与ERBD胆汁中肿瘤细胞的致瘤性及机制进行研究。以胆结石组作为阴性对照,分为良性结石组(30例),ERBD减黄组(13例),PTBD减黄组(14例),其中良性结石30例的胆汁中无肿瘤细胞,未形成肿瘤球,ERBD减黄13例胆汁中有3例形成肿瘤球(23%),PTBD减黄14例胆汁有6例形成肿瘤球(42%),PTBD组细胞的肿瘤球形成能力显著高于ERBD组。皮下移植瘤检测表明,PTBD组移植瘤生长能力显著高于ERBD组,PTBD胆汁中的肿瘤细胞具有更强的致瘤能力。在机制研究中,RT-PCR检测表明,PTBD组肿瘤球干细胞转录因子Nanog的mRNA水平显著高于ERBD组。在3例PTBD组细胞中敲低Nanog,肿瘤球形成,皮下移植瘤生长均被降低,Nanog在PTBD肿瘤细胞强的致瘤能力中发挥重要作用。PTBD细胞中Nanog的mRNA半衰期长于ERBD组,Nanog的mRNA受转录后修饰潜在调控。本文发现,PTBD肿瘤细胞中Nanog的m 6A水平高于ERBD组。对RNA修饰的甲基转移酶及去甲基化酶进行检测表明,ALKBH5(α-ketoglutarate dependent dioxygenase alkb homolog 5)的mRNA表达水平在PTBD组低于ERBD组,且与Nanog的m 6A水平显著相关。敲低ALKBH 5,Nanog的m 6A水平升高,而过表达ALKBH 5则下调Nanog的m 6A水平。双荧光素酶活性检测表明,ALKBH 5敲低显著增强荧光素酶活性,而ALKBH 5过表达则降低荧光素酶活性。进一步研究证实,敲低ALKBH 5,Nanog的mRNA和蛋白质水平均被上Hilar cholangiocarcinoma is insidious in onset and often causes obstructive jaundice due to bile stasis,leading to impaired liver function.For tumors with malignant obstructive jaundice,biliary drainage is often performed before surgery in clinical practice.Currently,the commonly used drainage methods are percutaneous transhepatic biliary drainage(PTBD)and endoscopic retrograde biliary drainage(ERBD),but there are controversies over the advantages and disadvantages of the two drainage methods.PTBD drainage can often lead to tumor implantation metastasis,but the underlying mechanism remains unclear.We detected tumor cells in PTBD and ERBD bile samples from hilar cholangiocarcinoma patients,subsequently explored their tumorigenicity and mechanisms through tumorsphere assay in vitro and xenograft tumor models in vivo.The experiments included benign gallstones group(30 cases)as a negative control,PTBD group(14 cases)and ERBD group(13 cases).Tumorsphere formation was identified in 3 cases(23%)among the 13 cases of ERBD group,in 6 cases(42%)among the 14 cases of PTBD group,but there were no tumor cells or formed tumorspheres in the 30 cases of benign gallstone group.The tumor sphere formation ability of cells in the PTBD group was significantly higher than that in ERBD group.Subcutaneous xenograft tumor assays showed that tumor growth in the PTBD group was significantly higher than that in the ERBD group.Tumor cells in PTBD bile possessed stronger tumorigenicity compared with the ERBD group.Mechanically,stem cell transcription factor Nanog mRNA levels were significantly higher in the PTBD group compared to the ERBD group.Both tumorsphere formation and xenograft tumor growth were reduced by Nanog knockdown in three cases of the PTBD group,indicating the important roles of Nanog in tumorigenicity of PTBD group tumor cells.The half-life of Nanog mRNA was longer in PTBD group cells than in ERBD group cells,suggesting potential post-transcriptional regulation on Nanog mRNA.The Nanog m 6A level was higher in PTBD group tumor

关 键 词：经皮经肝胆道引流 内镜下逆行胆管引流 肝门部胆管癌 α-酮戊二酸依赖的双加氧酶alkb家族同系物5 Nanog 

分 类 号：Q522[生物学—生物化学]