应用激光捕获显微切割技术分离肝癌石蜡组织标本中细菌及螺杆菌基因的检测

Isolate bacterium in paraffin-embedded tissues of hepatocellular carcinoma by laser capture microdissection technology and detection of Helicobacter species gene

目的对肝癌石蜡组织标本病理切片中发现的细菌进行分离鉴定. 方法 38例肝癌石蜡组织标本经聚合酶链反应(PCR)扩增螺杆菌16S rRNA,阳性标本病理切片后行石炭酸-碱性品红染色观察幽门螺杆菌(H.pylori),应用激光捕获显微切割技术(LCM)分离光镜下观察到的细菌,分离的细菌经PCR扩增螺杆菌16S rRNA,PCR产物进行测序及同源比较,阳性者再扩增H.pylori的特异基因[相对分子质量(Mr)为26×103蛋白,H.pylori种特异抗原]和相关功能基因(cagA, vacA). 结果15例肝癌石蜡组织标本中检测到螺杆菌16S rRNA,选取观察到细菌数量较多的6例用于LCM分离.分离的6例细菌样本均扩增出螺杆菌16S rRNA,经测序与H.pylori有99%～100%的同源性.4例Mr为26×103蛋白基因阳性,1例扩增出cagA基因,未扩增出vacA基因. 结论肝癌石蜡组织中经PCR检测到的H.pylori就是病理观察到的细菌.

Objective To isolate and identify the bacteria in paraffin embedded liver tissues of hepatocellular carcinoma (HCC). Methods Thirty eight paraffin-embedded tissue samples of HCC were examined by polymerase chain reaction (PCR) with Helicobacter-specific 16S rRNA primers. Histological sections of positive samples were stained by carbolic acid-basic fuchsin and the bacteria in sections were isolated by laser capture microdissection(LCM) technology. Helicobacter species 16S rRNA gene, H.pylori specific-antigen (M r 26×103) gene, cytotoxin-associated gene A(cagA), vacuolating toxin gene (vacA) were detected by PCR. Results Fifteen samples were positive in Helicobacter-specific 16S rRNA gene and 6 samples with more bacteria were selected to isolate the bacteria observed in sections by LCM. All six samples have been amplified for 16S rRNA (400 bp) by PCR, 4 samples were positive for M r 26×103, 1 positive for cagA and no positive in vacA. Three samples of 16S rRNA amplicons were sequenced and showed a 99%-100% identity to H.pylori. Conclusion H.pylori detected by PCR in liver tissues of HCC was the same one as the bacteria observed in histologic sections.