螺杆菌感染与肝癌关系的研究
彭小宁，范学工，黄燕，中南大学湘雅医院传染科 湖南省长沙市 410008
王志明，中南大学湘雅医院普外科 湖南省长沙市 410008
陈永平，中南大学湘雅医院病理科 湖南省长沙市 410008
彭小宁，男，1967-11-04生，湖南衡阳人，汉族，1995年衡阳医学院消化内科硕士研究生毕业，2001年湘雅医院传染病学博士研究生毕业，现在美国主要从事艾滋病方面的研究.
国家教育部优秀骨干教师基金(教技司2000-65)、湖南省自然科学基金(01jjy2114)和湖南省卫生厅科研基金(00022)资助
项目负责人 范学工，410008，湖南省长沙市，中南大学湘雅医院传染科. xgfan@hotmail.com
电话： 0731-4328926 传真： 0731-4327332
收稿日期 2002-01-16 接收日期 2002-02-07
The study on relationship between Helicobacter infection and primary liver carcinoma
Xiao-Ning Peng, Xue-Gong Fan, Yan Huang,Zhi-Ming Wang , Yong-Ping Cheng
Xiao-Ning Peng, Xue-Gong Fan, Yan Huang, Departments of Infectious Diseases of Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
Zhi-Ming Wang, Departments of Surgery of Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
Yong-Ping Cheng, Departments of Pathology of Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
Supported by the Splendid Teacher Foundation of the National Ministry of Education (No.2000-65) , Nature Scientific Foundation of Hunan Province (No. 01jjy2114) and the Scientific Research Foundation of Sanitary Government, Hunan Province (No. 00022)
Correspondence to: Prof. Xue-Gong Fan, Departments of Infectious Diseases, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China. xgfan@hotmail.com
Received 2002-01-16 Accepted 2002-02-07
AbstractAIM:To investigate the relationship between helicobacter species and primary hepatocellular carcinoma.
METHODS: Fifteen patients with primary liver carcinoma diagnosed by histopathology and 13 patients without as control were studied. Helicobacter species in liver specimens from studied subjects were examined by polymerase chain reaction (PCR) using helicobacter-specific 16S rRNA primers. Amplified products were identified by Southern blot and sequenced.
RESULTS: Helicobacter species were found in liver tissue from 60% (9/15) patients with primary liver carcinoma by PCR while none was found in control group (P < 0.01). Four of helicobacter-specific PCR amplicons were sequenced and the homology was 99% with 16S rRNA of H. pylori.
CONCLUSIONS:Helicobacter species are be present in the liver of patients with primary hepato cellular carcinoma and it is probably linked to carcinogenesis of liver, malignancy.
Peng XN, Fan XG, Huang Y, Wang ZM, Cheng YP. The study on relationship between helicobacter infection and primary liver carcinoma. Shijie Huaren Xiaohua Zazhi 2002;10(8):902-906
摘要
目的:已有研究发现几种螺杆菌与某些动物肝脏疾病相关，本研究目的旨在探讨螺杆菌感染是否与人类肝癌的发生相关.
方法:选取经病理诊断的肝癌患者15例作为研究对象，非肝癌患者13例作对照.用聚合酶链反应(PCR)扩增肝组织螺杆菌16S rRNA来检测螺杆菌，扩增产物用Southern杂交证实，并进行测序及同源比较.
结果:60%(9/15)肝癌患者肝组织中发现螺杆菌存在，而非肝癌组无1例阳性(P < 0.01)；所有PCR阳性产物用Southern杂交得到证实；4例测序及同源比较显示，肝癌组织中的螺杆菌与幽门螺杆菌有99%的同源性.
结论:肝癌患者肝组织存在螺杆菌感染，他与肝癌的发生可能存在某种联系.其是肝癌的致病因素抑或伴发感染，需进一步的研究确定.由于是扩增16S rRNA的部分序列，故目前仍不能明确是哪一种螺杆菌.
彭小宁,范学工, 黄燕, 王志明, 陈永平. 螺杆菌感染与肝癌关系的研究. 世界华人消化杂志 2002;10( 8):902-906
0 引言幽门螺杆菌(H. pylori)作为消化性溃疡、慢性胃炎、胃黏膜相关淋巴组织淋巴瘤、胃癌的重要致病因素已被实验研究和临床观察所证实[1-11].近年来，人们发现一些螺杆菌(Helicobacter)与某些动物的肝脏疾病密切相关，特别是肝螺杆菌(H. hepaticus)经动物实验证实能导致某些品系小鼠肝癌的发生[12,13].慢性乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)作为肝脏疾病和肝癌发生的最主要病因[14,15]，已有许多研究.螺杆菌感染是否也在人肝癌的发生中起作用，是一个令人十分感兴趣的课题.为此，笔者采用细菌培养、聚合酶链反应(PCR)、DNA序列分析、核酸杂交技术，检测了15例肝癌标本和13例非肝癌的肝脏标本螺杆菌，现报道如下.
1 材料和方法
1.1 材料 于2000-04-08在湘雅医院无菌操作收集28
例肝脏标本，并分为肝癌组(组Ⅰ)和非肝癌组(组Ⅱ)，所有对象均系我院外科住院患者.组Ⅰ 15例，经临床和病理诊断为原发性肝癌，其中男12例，女3例，年龄20~64岁，平均42.8岁.组Ⅱ 13例，男7例，女6例，年龄28岁~68岁，平均42.4岁，其中肝脏良性肿瘤2例，肝脏局灶性结节增生1例，余10例为胃部、胆道或结肠疾患(表1)，除1例有脂肪变性外，余9例肝组织均正常.组Ⅰv 15例肝癌患者中，14例HBV血清标志物(HBV-M)阳性；而组Ⅱ仅2例肝脏良性肿瘤抗-HBs阳性.
1.2 方法
1.2.1 肝组织标本的病理观察 15例肝癌、2例肝良性肿瘤和1例肝局灶性结节增生，系我院病理科的病理诊断.其余10例均为非肝脏疾病，将手术切取的少量肝脏标本，用10%的甲醛固定，石蜡包埋，用常规伊红—苏木素(H.E)染色，亦由我院病理科医生检查.
1.2.2 螺杆菌培养 按我室建立的H.pylori培养方法进行[16-18].将从手术室收集的新鲜肝癌标本冷藏，并立即运送到传染科细菌培养室，取少量肝癌组织在1ml磷酸缓冲盐(PBS，pH7.4)中用匀浆器制成匀浆，并种置于含7%羊血、10 g/L万古霉素、2 g/L二性霉素B、2.5 g/L多粘菌素B的哥伦比亚琼脂平板(Oxoid公司)，在微需氧环境即5%O2、10%CO2、85%N2，高湿度，37℃条件下培养，3d 后，每天观察有无菌落生长，共观察10 d .
1.2.3 DNA抽提 根据文献[19-21]报道方法并略作修改.取100mg肝组织置于冷的TE(pH7.5)缓冲液中，用匀浆器制成匀浆，5 000 r/min离心10 min，弃上清，加裂解液200μl(50mmol/L Tris-HCl，pH8.3，1mmol/L EDTA，0.75%Tween-20，300mg/L蛋白酶K)，55℃作用12~24 hr，95℃加热10 min灭活蛋白酶K，10 000 r/min离心5min，取上清分装并储存于-20℃备用.细菌DNA抽提采用溶菌酶、蛋白酶K及酚/氯仿法提取[22-24].
1.2.4 PCR扩增螺杆菌16S rRNA 采用文献[25,26]针对螺杆菌属16S rRNA设计的特异引物(为所有螺杆菌的通用引物)，C97：5’-GCT ATG ACG GGT ATC C-3’(nt276—291)，C98：5’-GAT TTT ACC CCT ACA CCA-3’(nt681—698)(上海生工生物技术公司合成)，扩增靶片段长度为400bp.PCR总反应体积为50μl，内含0.5μmol C97/C98引物，2.5U Taq DNA聚合酶，2.5mmol MgCl2，0.2mmol dNTP(以上试剂均为上海生工生物技术公司产品)，模板5-10μl.PCR反应条件为95℃预变性5min，冷却至70℃，加入Taq酶，进入扩增循环，94℃变性1min，55℃退火1.5min，72℃延伸2min，共扩增35个循环，终止延伸72℃10min.以幽门螺杆菌标准株ATCC49503 DNA作为阳性对照，去离子水作阴性对照.10-15μl PCR产物点样于1.5%琼脂糖凝胶电泳，0.5μg/ml溴化乙锭染色，于紫外灯下显影，并经UV-2000紫外分析仪(上海天能公司)照相.PCR仪为PE公司480型.
1.2.5 Southern杂交 为了证实扩增产物是细菌16S rRNA，而不是来源于人基因组的非特异性产物，我们用螺杆菌16S rDNA探针进行Southern杂交验证.用C97/C98引物扩增H. pylori标准株ATCC49503的DNA，产生400bp目的片段，经低熔点琼脂糖凝胶电泳后，用DNA纯化试剂盒(Gene公司)纯化，即制成所需探针，并测好浓度.用随机引物标记试剂盒(Worthington公司)，并按说明书用32P—dCTP(北京亚辉生物工程公司)标记探针.离心柱层析法纯化探针，用经典毛细管转移法将12μl PCR产物转移至硝酸纤维素膜(Amersham公司)上，80℃烤干1.5h，4℃保存备用.将硝酸纤维素膜预杂交3h，加入变性探针，过夜杂交后，洗膜，放射自显影48h.以幽门螺杆菌为阳性对照，去离子水作阴性对照.
1.2.6 测序及分析 将PCR产物直接测序，测序仪为ABI377，测序引物为C97和C98，采用正、反双向测序，由上海瑞真生物技术公司完成.在Genbank中用Blast程序进行同源比较.
统计学处理 实验结果以x2检验进行处理.
2 结果
2.1 细菌培养 培养15例肝癌及1例肝脏良性肿瘤标本，连续观察至培养的第10天，均无细菌生长.
2.2 肝脏组织病理学 详见表1.
表1 28例肝脏标本组织病理学
2.3 PCR扩增螺杆菌16S rRNA 用螺杆菌属特异引物C97/C98扩增螺肝菌16S rRNA，在15例肝癌标本中有9例扩增出400bp的目的片段 (表2， 图1)，阳性率为60%，而非肝癌组中没有1例阳性，二者差异有显著性(x2=11.49，P < 0.01).
2.4 Southern杂交 所有PCR阳性标本均用32P标记的从H.pylori标准株制备的16S rDNA探针，通过Southern杂交得到证实(图2).
表2 肝癌组病理诊断、病毒标志及PCR结果
1: 相对分子质量标准参照; 2: 阳性对照;
3: 阴性对照; 4: 空白对照; 5~9: 肝癌阳性标本;
图1 (PDF) 螺杆菌属特异引物PCR扩增肝脏标本16S rRNA结果
1~7: 肝癌组织阳性标本; 8: 阴性对照; 9: 阳性对照.
图2 (PDF) PCR产物Southern杂交结果
2.5 DNA序列分析 随机选取4个阳性标本的PCR产物进行序列分析，用C97和C98两条引物分别进行正向和反向测序，经拼接后进行同源比较，结果发现均与H.pylori 16S rRNA基因(基因登录号AF302106)具有99%的同源性.
3 讨论自1982年发现 H.pylori 以来，迄今为止已发现至少18种已正式命名的螺杆菌，其中有5种螺杆菌幼禽螺杆菌(H. pullorum)、肝螺杆菌、胆汁螺杆菌(H. bilis)、胆囊螺杆菌(H. cholecystus)、犬螺杆菌(H. canis)与某些动物肝脏疾病有明确的关系[27-34].继Fox et al [25]报道智利人慢性胆囊炎的胆囊黏膜与胆汁中有螺杆菌16S rRNA存在后，Nilson在原发性硬化性胆管炎和原发性胆汁性肝硬化患者的肝脏标本中发现螺杆菌16S rRNA存在[35].新近，Avenaud在8例肝癌标本中发现了螺杆菌16S rDNA存在，测序证明与H.pylori具有99%同源性[19].但他们均未能成功分离培养出螺杆菌.因而螺杆菌与人类肝脏疾病和肝癌的关系已渐成为这一领域新的热点.
本研究试图从肝脏标本中培养分离螺杆菌，令人遗憾的是，15例肝癌及1例肝良性肿瘤的培养均未成功.Fox和Avenaud [19,25]未能从冰冻组织中分离培养出螺杆菌，归咎于细菌在冰冻的组织中缺乏活力，我们从新鲜组织中仍未能成功培养出螺杆菌，可能的原因有：(1) 细菌的数量很少，培养不出来；(2) 细菌处于非可培养状态；(3) 肝内螺杆菌与H.pylori的生物学特性有差别，目前所使用的培养基和培养技术不适合这种微生物的生长.
近年来，随着分子生物学的发展，PCR技术已成为临床微生物检测的有力工具.例如海尔曼螺杆菌(H. heilmannii)和Whipple病的致病菌汉氏巴尔通氏体(Bartonella henselae)均是在这些细菌成功分离培养以前，用PCR方法发现的[36,37].16S rRNA基因因具有:(1)在生物进化中比其他基因演变得慢，因其保守性而被冠以细菌“分子化石”(molecule fossil)之称；(2)保守性是相对的，不同科、属、种间都有不同程度的差异；16S rRNA基因同源性在98.8%以上即可被认为是同一个种[38]；(3)不能够侧向转移；(4)大、小适度(1600nt)，能满足进行比较的要求.因而,通过PCR扩增16S rRNA，然后进行序列分析，将所得的16S rRNA序列与已知的16S rRNA序列进行比较，可以对未知细菌进行基本的鉴定和分类[39,40].
用螺杆菌16S rRNA的特异引物，经PCR方法对15例肝癌组织和13例非肝癌的肝脏组织进行螺杆菌16S rRNA基因检测，结果60%(9/15)的肝脏组织中发现螺杆菌存在，PCR产物进一步通过Southern杂交证实.证明肝癌患者肝组织确有螺杆菌感染，其阳性率接近于法国学者Avenaud et al [19]新近报道.对照组未发现1例阳性，两组之间差异显著.尽管目前尚难以下肯定结论，但由于非肝癌组肝组织中未发现1例螺杆菌阳性标本，因而笔者认为，螺杆菌感染与肝癌的发生存在某种联系.Avenaud选取的肝癌标本均没有肝硬化，也没有肝炎病毒感染，并推测有可能类似肝螺杆菌的致癌机制(一个重要特点是无肝硬化过程).在我国80%左右肝癌的发生与HBV慢性感染密切相关，大多经历肝硬化过程.本文报道的9例螺杆菌阳性肝癌标本，6例有HBV现症感染，4例伴有肝硬化，3例没有HBV现症感染而螺杆菌阳性，因而螺杆菌阳性感染是仅仅作为一个伴随感染还是与肝炎病毒一道参与肝癌的发生，现尚难以确定.进一步的工作本研究组正在进行之中.
选取4个标本的PCR产物，通过测序，经同源比较，发现与H.pylori有99%的同源性.但由于是螺杆菌的16S rRNA部分序列(400bp)，我们还不能确定就是H.pylori，有待进一步明确.
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40 Relman DA. Detecion and identification of previously unrecognized microbial pathogens. Emerg Infect Dis 1998;4:490-495, http://www.100md.com(彭小宁，范学工，黄 燕，王志明，陈永平)
王志明，中南大学湘雅医院普外科 湖南省长沙市 410008
陈永平，中南大学湘雅医院病理科 湖南省长沙市 410008
彭小宁，男，1967-11-04生，湖南衡阳人，汉族，1995年衡阳医学院消化内科硕士研究生毕业，2001年湘雅医院传染病学博士研究生毕业，现在美国主要从事艾滋病方面的研究.
国家教育部优秀骨干教师基金(教技司2000-65)、湖南省自然科学基金(01jjy2114)和湖南省卫生厅科研基金(00022)资助
项目负责人 范学工，410008，湖南省长沙市，中南大学湘雅医院传染科. xgfan@hotmail.com
电话： 0731-4328926 传真： 0731-4327332
收稿日期 2002-01-16 接收日期 2002-02-07
The study on relationship between Helicobacter infection and primary liver carcinoma
Xiao-Ning Peng, Xue-Gong Fan, Yan Huang,Zhi-Ming Wang , Yong-Ping Cheng
Xiao-Ning Peng, Xue-Gong Fan, Yan Huang, Departments of Infectious Diseases of Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
Zhi-Ming Wang, Departments of Surgery of Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
Yong-Ping Cheng, Departments of Pathology of Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
Supported by the Splendid Teacher Foundation of the National Ministry of Education (No.2000-65) , Nature Scientific Foundation of Hunan Province (No. 01jjy2114) and the Scientific Research Foundation of Sanitary Government, Hunan Province (No. 00022)
Correspondence to: Prof. Xue-Gong Fan, Departments of Infectious Diseases, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China. xgfan@hotmail.com
Received 2002-01-16 Accepted 2002-02-07
AbstractAIM:To investigate the relationship between helicobacter species and primary hepatocellular carcinoma.
METHODS: Fifteen patients with primary liver carcinoma diagnosed by histopathology and 13 patients without as control were studied. Helicobacter species in liver specimens from studied subjects were examined by polymerase chain reaction (PCR) using helicobacter-specific 16S rRNA primers. Amplified products were identified by Southern blot and sequenced.
RESULTS: Helicobacter species were found in liver tissue from 60% (9/15) patients with primary liver carcinoma by PCR while none was found in control group (P < 0.01). Four of helicobacter-specific PCR amplicons were sequenced and the homology was 99% with 16S rRNA of H. pylori.
CONCLUSIONS:Helicobacter species are be present in the liver of patients with primary hepato cellular carcinoma and it is probably linked to carcinogenesis of liver, malignancy.
Peng XN, Fan XG, Huang Y, Wang ZM, Cheng YP. The study on relationship between helicobacter infection and primary liver carcinoma. Shijie Huaren Xiaohua Zazhi 2002;10(8):902-906
摘要
目的:已有研究发现几种螺杆菌与某些动物肝脏疾病相关，本研究目的旨在探讨螺杆菌感染是否与人类肝癌的发生相关.
方法:选取经病理诊断的肝癌患者15例作为研究对象，非肝癌患者13例作对照.用聚合酶链反应(PCR)扩增肝组织螺杆菌16S rRNA来检测螺杆菌，扩增产物用Southern杂交证实，并进行测序及同源比较.
结果:60%(9/15)肝癌患者肝组织中发现螺杆菌存在，而非肝癌组无1例阳性(P < 0.01)；所有PCR阳性产物用Southern杂交得到证实；4例测序及同源比较显示，肝癌组织中的螺杆菌与幽门螺杆菌有99%的同源性.
结论:肝癌患者肝组织存在螺杆菌感染，他与肝癌的发生可能存在某种联系.其是肝癌的致病因素抑或伴发感染，需进一步的研究确定.由于是扩增16S rRNA的部分序列，故目前仍不能明确是哪一种螺杆菌.
彭小宁,范学工, 黄燕, 王志明, 陈永平. 螺杆菌感染与肝癌关系的研究. 世界华人消化杂志 2002;10( 8):902-906
0 引言幽门螺杆菌(H. pylori)作为消化性溃疡、慢性胃炎、胃黏膜相关淋巴组织淋巴瘤、胃癌的重要致病因素已被实验研究和临床观察所证实[1-11].近年来，人们发现一些螺杆菌(Helicobacter)与某些动物的肝脏疾病密切相关，特别是肝螺杆菌(H. hepaticus)经动物实验证实能导致某些品系小鼠肝癌的发生[12,13].慢性乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)作为肝脏疾病和肝癌发生的最主要病因[14,15]，已有许多研究.螺杆菌感染是否也在人肝癌的发生中起作用，是一个令人十分感兴趣的课题.为此，笔者采用细菌培养、聚合酶链反应(PCR)、DNA序列分析、核酸杂交技术，检测了15例肝癌标本和13例非肝癌的肝脏标本螺杆菌，现报道如下.
1 材料和方法
1.1 材料 于2000-04-08在湘雅医院无菌操作收集28
例肝脏标本，并分为肝癌组(组Ⅰ)和非肝癌组(组Ⅱ)，所有对象均系我院外科住院患者.组Ⅰ 15例，经临床和病理诊断为原发性肝癌，其中男12例，女3例，年龄20~64岁，平均42.8岁.组Ⅱ 13例，男7例，女6例，年龄28岁~68岁，平均42.4岁，其中肝脏良性肿瘤2例，肝脏局灶性结节增生1例，余10例为胃部、胆道或结肠疾患(表1)，除1例有脂肪变性外，余9例肝组织均正常.组Ⅰv 15例肝癌患者中，14例HBV血清标志物(HBV-M)阳性；而组Ⅱ仅2例肝脏良性肿瘤抗-HBs阳性.
1.2 方法
1.2.1 肝组织标本的病理观察 15例肝癌、2例肝良性肿瘤和1例肝局灶性结节增生，系我院病理科的病理诊断.其余10例均为非肝脏疾病，将手术切取的少量肝脏标本，用10%的甲醛固定，石蜡包埋，用常规伊红—苏木素(H.E)染色，亦由我院病理科医生检查.
1.2.2 螺杆菌培养 按我室建立的H.pylori培养方法进行[16-18].将从手术室收集的新鲜肝癌标本冷藏，并立即运送到传染科细菌培养室，取少量肝癌组织在1ml磷酸缓冲盐(PBS，pH7.4)中用匀浆器制成匀浆，并种置于含7%羊血、10 g/L万古霉素、2 g/L二性霉素B、2.5 g/L多粘菌素B的哥伦比亚琼脂平板(Oxoid公司)，在微需氧环境即5%O2、10%CO2、85%N2，高湿度，37℃条件下培养，3d 后，每天观察有无菌落生长，共观察10 d .
1.2.3 DNA抽提 根据文献[19-21]报道方法并略作修改.取100mg肝组织置于冷的TE(pH7.5)缓冲液中，用匀浆器制成匀浆，5 000 r/min离心10 min，弃上清，加裂解液200μl(50mmol/L Tris-HCl，pH8.3，1mmol/L EDTA，0.75%Tween-20，300mg/L蛋白酶K)，55℃作用12~24 hr，95℃加热10 min灭活蛋白酶K，10 000 r/min离心5min，取上清分装并储存于-20℃备用.细菌DNA抽提采用溶菌酶、蛋白酶K及酚/氯仿法提取[22-24].
1.2.4 PCR扩增螺杆菌16S rRNA 采用文献[25,26]针对螺杆菌属16S rRNA设计的特异引物(为所有螺杆菌的通用引物)，C97：5’-GCT ATG ACG GGT ATC C-3’(nt276—291)，C98：5’-GAT TTT ACC CCT ACA CCA-3’(nt681—698)(上海生工生物技术公司合成)，扩增靶片段长度为400bp.PCR总反应体积为50μl，内含0.5μmol C97/C98引物，2.5U Taq DNA聚合酶，2.5mmol MgCl2，0.2mmol dNTP(以上试剂均为上海生工生物技术公司产品)，模板5-10μl.PCR反应条件为95℃预变性5min，冷却至70℃，加入Taq酶，进入扩增循环，94℃变性1min，55℃退火1.5min，72℃延伸2min，共扩增35个循环，终止延伸72℃10min.以幽门螺杆菌标准株ATCC49503 DNA作为阳性对照，去离子水作阴性对照.10-15μl PCR产物点样于1.5%琼脂糖凝胶电泳，0.5μg/ml溴化乙锭染色，于紫外灯下显影，并经UV-2000紫外分析仪(上海天能公司)照相.PCR仪为PE公司480型.
1.2.5 Southern杂交 为了证实扩增产物是细菌16S rRNA，而不是来源于人基因组的非特异性产物，我们用螺杆菌16S rDNA探针进行Southern杂交验证.用C97/C98引物扩增H. pylori标准株ATCC49503的DNA，产生400bp目的片段，经低熔点琼脂糖凝胶电泳后，用DNA纯化试剂盒(Gene公司)纯化，即制成所需探针，并测好浓度.用随机引物标记试剂盒(Worthington公司)，并按说明书用32P—dCTP(北京亚辉生物工程公司)标记探针.离心柱层析法纯化探针，用经典毛细管转移法将12μl PCR产物转移至硝酸纤维素膜(Amersham公司)上，80℃烤干1.5h，4℃保存备用.将硝酸纤维素膜预杂交3h，加入变性探针，过夜杂交后，洗膜，放射自显影48h.以幽门螺杆菌为阳性对照，去离子水作阴性对照.
1.2.6 测序及分析 将PCR产物直接测序，测序仪为ABI377，测序引物为C97和C98，采用正、反双向测序，由上海瑞真生物技术公司完成.在Genbank中用Blast程序进行同源比较.
统计学处理 实验结果以x2检验进行处理.
2 结果
2.1 细菌培养 培养15例肝癌及1例肝脏良性肿瘤标本，连续观察至培养的第10天，均无细菌生长.
2.2 肝脏组织病理学 详见表1.
表1 28例肝脏标本组织病理学
| n | 组织病理(例数) | |
| 肝癌组(组I) | 15 | 原发性肝细胞性肝癌(14)，其中9例伴有肝硬化胆管上皮细胞型肝癌(1) |
| 非肝癌组(组II) | 13 | 肝细胞腺瘤(1);肝海绵状血管瘤(1);肝局灶性结节增生(1);胃部疾病肝脏标本(3):肝小叶结构正常,可见少量淋巴细胞浸润,无明显的纤维组织增生,其中有1例脂肪变性;慢性胆囊疾病(5):肝小叶结构清晰,部分有轻度纤维组织增生,可见少量炎症细胞浸润,其中1例有寄生虫感染;结肠癌(1):肝小叶结构正常,少量纤维组织增生及炎症细胞浸润,胆管增生;胆总管癌(1):肝小叶结构正常,胆管扩张,肝窦及小血管扩张、充血,有炎症细胞浸润. |
2.3 PCR扩增螺杆菌16S rRNA 用螺杆菌属特异引物C97/C98扩增螺肝菌16S rRNA，在15例肝癌标本中有9例扩增出400bp的目的片段 (表2， 图1)，阳性率为60%，而非肝癌组中没有1例阳性，二者差异有显著性(x2=11.49，P < 0.01).
2.4 Southern杂交 所有PCR阳性标本均用32P标记的从H.pylori标准株制备的16S rDNA探针，通过Southern杂交得到证实(图2).
表2 肝癌组病理诊断、病毒标志及PCR结果
| 患者 | 性别 | 年龄(岁) | 病理诊断 | HBV-M | 抗-HCV | PCR |
| 1 | 女 | 63 | 肝高分化腺癌 | - | - | + |
| 2 | 男 | 50 | 原发性肝细胞性肝癌 | sAg+, eAb+, cAb+ | 未做 | - |
| 门脉性肝硬化 | ||||||
| 3 | 女 | 20 | 原发性肝细胞性肝癌 | sAg+, eAb+, cAb+ | - | - |
| 4 | 男 | 37 | 原发性肝细胞性肝癌 | sAg+, cAb+ | - | + |
| 5 | 男 | 42 | 原发性肝细胞性肝癌 | sAg+, eAb+, cAb+ | - | + |
| 6 | 男 | 37 | 原发性肝细胞性肝癌 | sAg+, cAb+ | 未做 | + |
| 门脉性肝硬化 | ||||||
| 7 | 男 | 64 | 原发性肝癌 | sAb+ | 未做 | + |
| (胆管上皮型) | ||||||
| 8 | 男 | 35 | 原发性肝细胞性肝癌 | eAb+ | 未做 | + |
| 9 | 男 | 58 | 原发性肝细胞性肝癌 | sAg+, eAg+, cAb+ | - | + |
| 门脉性肝硬化 | ||||||
| 10 | 男 | 42 | 原发性肝细胞性肝癌 | sAg+, eAg+, cAb+ | 未做 | + |
| 门脉性肝硬化 | ||||||
| 11 | 女 | 33 | 原发性肝细胞性肝癌 | sAg+, cAb+ | 未做 | + |
| 门脉性肝硬化 | ||||||
| 12 | 男 | 50 | 原发性肝细胞性肝癌 | sAg+, eAb+, cAb+ | - | - |
| 门脉性肝硬化 | ||||||
| 13 | 男 | 34 | 原发性肝细胞性肝癌 | sAg+, cAb+ | - | - |
| 门脉性肝硬化 | ||||||
| 14 | 男 | 28 | 原发性肝细胞性肝癌 | sAg+, eAb+, cAb+ | - | - |
| 门脉性肝硬化 | ||||||
| 15 | 男 | 50 | 原发性肝细胞性肝癌 | sAg+, cAb+ | - | - |
| 门脉性肝硬化 |
1: 相对分子质量标准参照; 2: 阳性对照;
3: 阴性对照; 4: 空白对照; 5~9: 肝癌阳性标本;
图1 (PDF) 螺杆菌属特异引物PCR扩增肝脏标本16S rRNA结果
1~7: 肝癌组织阳性标本; 8: 阴性对照; 9: 阳性对照.
图2 (PDF) PCR产物Southern杂交结果
2.5 DNA序列分析 随机选取4个阳性标本的PCR产物进行序列分析，用C97和C98两条引物分别进行正向和反向测序，经拼接后进行同源比较，结果发现均与H.pylori 16S rRNA基因(基因登录号AF302106)具有99%的同源性.
3 讨论自1982年发现 H.pylori 以来，迄今为止已发现至少18种已正式命名的螺杆菌，其中有5种螺杆菌幼禽螺杆菌(H. pullorum)、肝螺杆菌、胆汁螺杆菌(H. bilis)、胆囊螺杆菌(H. cholecystus)、犬螺杆菌(H. canis)与某些动物肝脏疾病有明确的关系[27-34].继Fox et al [25]报道智利人慢性胆囊炎的胆囊黏膜与胆汁中有螺杆菌16S rRNA存在后，Nilson在原发性硬化性胆管炎和原发性胆汁性肝硬化患者的肝脏标本中发现螺杆菌16S rRNA存在[35].新近，Avenaud在8例肝癌标本中发现了螺杆菌16S rDNA存在，测序证明与H.pylori具有99%同源性[19].但他们均未能成功分离培养出螺杆菌.因而螺杆菌与人类肝脏疾病和肝癌的关系已渐成为这一领域新的热点.
本研究试图从肝脏标本中培养分离螺杆菌，令人遗憾的是，15例肝癌及1例肝良性肿瘤的培养均未成功.Fox和Avenaud [19,25]未能从冰冻组织中分离培养出螺杆菌，归咎于细菌在冰冻的组织中缺乏活力，我们从新鲜组织中仍未能成功培养出螺杆菌，可能的原因有：(1) 细菌的数量很少，培养不出来；(2) 细菌处于非可培养状态；(3) 肝内螺杆菌与H.pylori的生物学特性有差别，目前所使用的培养基和培养技术不适合这种微生物的生长.
近年来，随着分子生物学的发展，PCR技术已成为临床微生物检测的有力工具.例如海尔曼螺杆菌(H. heilmannii)和Whipple病的致病菌汉氏巴尔通氏体(Bartonella henselae)均是在这些细菌成功分离培养以前，用PCR方法发现的[36,37].16S rRNA基因因具有:(1)在生物进化中比其他基因演变得慢，因其保守性而被冠以细菌“分子化石”(molecule fossil)之称；(2)保守性是相对的，不同科、属、种间都有不同程度的差异；16S rRNA基因同源性在98.8%以上即可被认为是同一个种[38]；(3)不能够侧向转移；(4)大、小适度(1600nt)，能满足进行比较的要求.因而,通过PCR扩增16S rRNA，然后进行序列分析，将所得的16S rRNA序列与已知的16S rRNA序列进行比较，可以对未知细菌进行基本的鉴定和分类[39,40].
用螺杆菌16S rRNA的特异引物，经PCR方法对15例肝癌组织和13例非肝癌的肝脏组织进行螺杆菌16S rRNA基因检测，结果60%(9/15)的肝脏组织中发现螺杆菌存在，PCR产物进一步通过Southern杂交证实.证明肝癌患者肝组织确有螺杆菌感染，其阳性率接近于法国学者Avenaud et al [19]新近报道.对照组未发现1例阳性，两组之间差异显著.尽管目前尚难以下肯定结论，但由于非肝癌组肝组织中未发现1例螺杆菌阳性标本，因而笔者认为，螺杆菌感染与肝癌的发生存在某种联系.Avenaud选取的肝癌标本均没有肝硬化，也没有肝炎病毒感染，并推测有可能类似肝螺杆菌的致癌机制(一个重要特点是无肝硬化过程).在我国80%左右肝癌的发生与HBV慢性感染密切相关，大多经历肝硬化过程.本文报道的9例螺杆菌阳性肝癌标本，6例有HBV现症感染，4例伴有肝硬化，3例没有HBV现症感染而螺杆菌阳性，因而螺杆菌阳性感染是仅仅作为一个伴随感染还是与肝炎病毒一道参与肝癌的发生，现尚难以确定.进一步的工作本研究组正在进行之中.
选取4个标本的PCR产物，通过测序，经同源比较，发现与H.pylori有99%的同源性.但由于是螺杆菌的16S rRNA部分序列(400bp)，我们还不能确定就是H.pylori，有待进一步明确.
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