鼻咽癌患者不同部位EBV毒株基因多态性的检测与分析

发布时间：2018-05-30 16:55

本文选题：疱疹病毒4型 + 人　；参考：《青岛大学》2012年硕士论文

【摘要】：目的：应用PCR技术检测同一鼻咽癌患者配对的癌组织、咽漱液和外周血单个核细胞(PBMC)标本基因分型及序列分析是否一致,旨在说明同一鼻咽癌患者不同部位的EBV是否由同一种EBV毒株感染,以寻找是否存在高致癌性的EBV毒株。方法：选取20例临床确诊的鼻咽癌患者作为研究对象,取其石蜡包埋组织进行病理切片,采用原位杂交技术检测鼻咽癌组织中EBER1的转录表达,筛选鉴定出EBV阳性NPC患者。收集EBV阳性患者配对的石蜡包埋组织、咽漱液和PBMC提取DNA,应用PCR技术检测EBV1/2分型,PCR结合限制性片段长度多态性(RFLP)分析检测F/f分型以及C/D分型,同时对EBER和EBNA1基因扩增进行序列分析。结果：①20例鼻咽癌组织标本中有18例EBER1阳性,阳性率为90%。②18例阳性标本中有17例分型成功,1例患者癌组织和咽漱液配对标本1/2分型和F/f分型不同,其余配对标本的EBV基因分型均一致,同时以1型、F型及C型为主。③在18例配对标本的EBER和EBNA1基因序列分析中,有7例标本EBER和EBNA1基因扩增成功,其中2例患者癌组织和咽漱液配对标本在EBER基因,1例在EBNA1基因分析中表现为不同的序列变异,其余患者配对的癌组织和咽漱液中表现为相同序列,即同一患者癌组织和咽漱液是由同一种EBV毒株感染所致。④由于血液标本较难扩增,同一患者咽漱液、癌组织和PBMC配对标本仅有3例PCR扩增成功,其中1例在EBER基因2例在EBNA1基因分析中三种不同部位来源的EBV基因序列一致。结论：同一患者不同部位的基因分型和基因序列一致,表明同一患者不同部位的EBV由同一种EBV毒株感染。临床咽漱液标本的采集更为方便,提示鼻咽癌患者咽漱液中的EBV分型对于分析组织中的EBV毒株具有一定的指导意义。但是由于在个别患者中仍存在不一致的情况,因此在实验分析过程中应谨慎操作。
[Abstract]:Objective: to detect the consistency of genotyping and sequence analysis of PBMCs from matched NPC tissues, pharyngeal gargle and peripheral blood mononuclear cells (PBMCs) by PCR technique. The aim of this study was to find out whether the EBV in different parts of the same NPC patient was infected by the same EBV strain, and to find out if there was a high carcinogenic EBV strain. Methods: twenty patients with nasopharyngeal carcinoma (NPC) were selected as the study objects. The paraffin embedded tissues were selected for pathological sections. The expression of EBER1 in nasopharyngeal carcinoma was detected by in situ hybridization. The EBV positive NPC patients were screened out. The paraffin embedded tissues of EBV positive patients were collected. The DNA was extracted from pharynx gargle and PBMC. PCR technique was used to detect EBV1/2 typing and restriction fragment length polymorphism (RFLP) analysis to detect F / F typing and C / D typing. At the same time, EBER and EBNA1 gene amplification were sequenced. Results 18 out of 120 nasopharyngeal carcinoma tissues were positive for EBER1, and 17 of 90.218 positive specimens were classified successfully. One patient's carcinoma tissue and pharyngeal gargle matched samples were divided into 1 / 2 and F / f types. The EBV genotyping of the other matched samples was consistent. In the 18 matched samples, EBER and EBNA1 genes were amplified successfully in 7 out of 18 matched samples. Two cases of cancer tissue and pharyngeal gargle paired specimen showed different sequence variation in EBNA1 gene analysis in 1 case of EBER gene analysis, and the same sequence of cancer tissue and pharyngeal gargle in other cases. That is to say, the cancer tissue of the same patient and the pharyngeal gargle were caused by the same EBV strain infection. 4. The blood sample was difficult to be amplified. Only 3 cases of the same patient's pharyngeal gargle, cancer tissue and PBMC matched specimen were amplified successfully by PCR amplification. Among them, 1 case was in EBER gene, 2 cases were in EBNA1 gene analysis, the sequence of EBV gene from three different parts was the same. Conclusion: the genotyping and gene sequence of different parts of the same patient were the same, which indicated that the EBV of different parts of the same patient was infected by the same EBV strain. The collection of clinical pharyngeal gargle samples is more convenient, suggesting that EBV typing in nasopharyngeal carcinoma patients' pharyngeal gargle has certain guiding significance for the analysis of EBV strains in tissues. However, inconsistency still exists in individual patients, so caution should be exercised in the course of experimental analysis.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

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