原发性肝细胞癌病人肝组织中螺杆菌的检测及分析  

Detection and analysis of Helicobacter species in liver tissue from patients with primary hepatocellular carcinoma

作　　者：陈燕凌[1] 叶敬旻[1] 佘菲菲[2] 唐南洪[1] 李秀金[1] 王晓茜[1] 林旭[2] 

机构地区：[1]福建医科大学附属协和医院肝胆外科,福州市350001 [2]福建医科大学分子医学研究中心、福建省高校感染与肿瘤重点实验室

出　　处：《中华肝胆外科杂志》2007年第10期667-671,共5页Chinese Journal of Hepatobiliary Surgery

基　　金：本课题受福建省重大科技基金（基金编号：2002F005）及福建大学教授学术发展基金（JS06101）资助

摘　　要：目的检测并分析原发性肝细胞癌病人组织中螺杆菌的存在情况。方法取配对的80对肝癌、癌旁组织标本及13例正常肝组织标本，抽提组织DNA，分别以细菌特异性和螺杆菌属细菌特异性16SrRNA基因引物进行巢式PCR检测，RCR产物T-A克隆后进行测序和基因进化树分析；对16SrRNA基因归属于幽门螺杆菌的组织样品，PCR检测幽门螺杆菌特异性26KDa基因及cagA、vacA、rps4、g1mM四个功能相关基因，进一步验证幽门螺杆菌的存在；应用抗幽门螺杆菌抗体对肝癌／癌旁组织及正常肝标本进行免疫组化验测。结果39份肝癌组织标本（39／80，48．75％）及37份癌旁组织标本（37／80，46．25％）检出螺杆菌属16SrRNA基因，其中，肝癌组织及癌旁组织均阳性的共有20例，正常肝组织无一例阳性（P〈0．01）。对其中50份16SrRNA基因的进化树分析提示它们与幽门螺杆菌的同源性超过99％，与此相关的组织样本中，幽门螺杆菌特异性26KDa基因阳性37份，cagA基因阳性8份，g1mM基因阳性9份，vacA基因以及rps4基因未检出。免疫组化显示26份癌组织（26／80，32．5％）及24份癌旁组织（24／80，30．0％）检出幽门螺杆菌，且主要分布于肝窦、汇管区及小肝管，正常肝组织无一例阳性（P〈0．01）。结论原发性肝细胞癌病人的癌组织及（或）相应的癌旁组织内存在螺杆菌，螺杆菌感染与原发性肝癌发生可能存在某种关联。Objective To detect and analyze the Helieobaeter species in liver tissue from patients with primary hepatocellular carcinoma （HCC）. Methods Eighty paired tumor and peri-tumor tissues from the patients with primary HCC and 13 normal liver tissues were selected. The tissue DNA was extracted and subjected to the nested PCR using eubaeterial and Helicobaeter-speeifie 16SrRNA primers. The amplified DNA fragments were sequenced and analyzed after the T-A cloning. For the samples in which the 16SrDNA was categorized to Helicobacer pylori, further PCR was performed to detect 5 of the H. pylori-specific genes （i. e. 26 KDa, cagA, vacA, rsp4 and glmm）. Meanwhile, the immunohistochemical staining was used to ascertain the distribution of Hp in liver tissue. Results Thirty-nine out of the 80 （48.75%） tumor tissues and 37 out of the 80 （47.5%） peritumor tissues were positive for Helicobacter-specific 16SrRNA gene. The positive detection of the gene was found in 20 and but not in normal liver tissues （P〈0.01）. Sequence analysis of 50 PCR products revealed that they were related most closely to the 16SrDNA sequence of H. pylori （〉99%）. Furthermore, amongst these samples, 37 were positive for 26-KDa gene of H. pylori, 8 for cagA, 9 for glmM but none for vacA and rps4. Immunohistochemical staining indicated that 26 tumor tissues and 24 peri-tumor tissues were positive for H. pylori and the positive distribution was mainly found in hepatic sinusoid, bile canaliculi and portal area. However, it was negative in all the normal liver tissues （P〈0.01）. Conclusions Helieobaeter species can be detected in the tumor and peri-tumor tissues from the patients with HCC, which suggests that the infection of Helicobacter species is associated with carcinogenesis of HCC.

关 键 词：癌 肝细胞 聚合酶链反应 幽门螺杆菌 免疫组化 

分 类 号：R686[医药卫生—骨科学]