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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 found in the pathological sections of the liver cancer paraffin tissue specimens. Methods Thirty-eight paraffin-embedded specimens of HCC were amplified by polymerase chain reaction (PCR) from 16S rRNA of Helicobacter pylori. The pathological sections of positive specimens were stained with carnitine basic fuchsine for H.pylori infection. Laser capture microscopy Bacteria were isolated by light microscopy with cutting technique (LCM). The isolated bacteria were sequenced and homologous compared by PCR amplification of Helicobacter pylori 16S rRNA. Positive samples were re-amplified by H.pylori specific gene [relative molecular mass (Mr) is 26 × 103 protein, H. pylori species specific antigen] and related functional genes (cagA, vacA). Results 16 Helicobacter pylori (16S rRNA) were detected in 15 cases of hepatocellular carcinoma. Six cases with more bacteria were selected for LCM isolation. The 16S rRNA of Helicobacter was amplified from 6 bacterial samples isolated and sequenced. The sequence was homologous to H.pylori by 99% -100%. 4 cases Mr positive for 26 × 103 protein gene, cagA gene was amplified in 1 case, vacA gene was not amplified. Conclusions H. pylori detected by PCR in paraffin-embedded liver cancer is a pathologically observed bacterium.