食管鳞癌组织中幽门螺杆菌L型和线粒体DNA微卫星不稳定的检测及意义

Detection and Significance of Helicobacter pylori L Type and Mitochondrial DNA Microsatellite Instability in Esophageal Squamous Cell Carcinoma

目的:探讨幽门螺杆菌感染、线粒体DNA微卫星不稳定性与食管鳞状细胞癌的关系。方法:运用Giemsa染色方法,光镜下观察食管鳞状细胞癌组织中幽门螺杆菌L型形态特征;结合免疫组化染色及幽门螺杆菌尿素酶基因片段PCR扩增,检测食管鳞状细胞癌组织及正常对照组织中幽门螺杆菌L型感染率。用长片段PCR扩增食管鳞状细胞癌组织及正常对照组织线粒体DNA,并以此为模板,PCR扩增其控制区D-loop区微卫星位点(C)n序列和(CA)n序列,运用PCR-SSCP方法检测PCR产物微卫星不稳定情况。结果:1)食管鳞状细胞癌组织中,幽门螺杆菌感染率为65.2%(30/46),与正常组织比较有显著性差异。食管鳞状细胞癌组织中幽门螺杆菌形态主要为L型,分布于癌巢内及癌旁。幽门螺杆菌L型感染与食管鳞状细胞癌密切相关,并且与癌组织浸润深度和淋巴结转移具有相关性,与其它临床病理参数无相关性。2)46例食管鳞状细胞癌中,mtMSI检出率为32.6%(15/46),其中13例于D-loop区(C)n序列检出MSI,5例于D-loop区(CA)n序列检出MSI,于(C)n和(CA)n序列同时检出者3例,正常组织中未检出mtMSI,mtMSI与临床病理参数无关。3)46例食管鳞状细胞癌组织中,30例幽门螺杆菌感染阳性者有14例检出mtMSI,16例幽门螺杆菌阴性者有1例检出mtMSI。幽门螺杆菌感染阳性者mtMSI的检出率与幽门螺杆菌感染阴性者mtMSI的检出率之间有显著性差异。结论:线粒体DNA D-loop区(C)n序列微卫星不稳定性可能在食管鳞状细胞癌发生中起重要作用,并与幽门螺杆菌L型感染有关。

Objective： To investigate the correlation of Helicobacter pylori L type （Hp-L） and mitochondrial DNA microsatellite instability （mtMSI） with esophageal squamous cell carcinoma （ESCC）. Methods： Using Giemsa staining, immunohistochemistry （IHC） staining and polymerase chain reaction （PCR）, we detected Hp-L infection rate in 46 samples of ESCC and corresponding normal tissues at the incision margin. PCR technique was used to amplify the urase gene fragment of Hp. Hp-L morphological characters were observed by Giemsa staining and light microscope. The mitochondrial DNA （mtDNA） was amplified by Genepha Long PCR Kit. PCR product was regarded as the template. （C）n and （CA）n locus that existed in the D-loop zone of template mtDNA were amplified. The PCR product MSI was analyzed by PCR-SSCP technique. Results： The Hp infection rate was 65.2% （30/46） in ESCC, higher than that in normal control tissues. The main shape of Hp was Hp-L type which distributed into or beside nests of carcinoma. A correlation was found between Hp-L infection and ESCC. The invasive depth of tumor tissues and the regional lymph node metastasis were correlated with Hp-L infection, mtMSI detection rate was 32.6% （15/ 46） in 46 case of ESCC, of which 13 cases were examined at （C）n locus of D-loop zone, 5 cases were examined at （CA）n locus, and 3 cases were examined both at （C）n and （CA）n. mtMSI was not found in nomal tissues at the incision margin. No significant relationship was found between mtMSI and clinicopathologic charecteristics. The mtMSI incidence in the 30 cases of ESCC with Hp-L infection was 46.7% （14/30）. However, the mtMSI incidence in the 16 cases of ESCC without Hp-L infection was 6.25% （1/16）. The difference in the mtMSI detection rate was significant between ESCC with Hp-L infection and ESCC without Hp-L infection. Conclusion： MSl of mitochondrial D-loop zone （C）n locus may play an important role in the carcinogenesis and development of ESCC, and is correlated with Hp-L infection.