本文選題：皰疹病毒4型 + 人��； 參考：《青島大學(xué)》2012年碩士論文

【摘要】：目的：應(yīng)用PCR技術(shù)檢測(cè)同一鼻咽癌患者配對(duì)的癌組織、咽漱液和外周血單個(gè)核細(xì)胞(PBMC)標(biāo)本基因分型及序列分析是否一致,旨在說(shuō)明同一鼻咽癌患者不同部位的EBV是否由同一種EBV毒株感染,以尋找是否存在高致癌性的EBV毒株。方法：選取20例臨床確診的鼻咽癌患者作為研究對(duì)象,取其石蠟包埋組織進(jìn)行病理切片,采用原位雜交技術(shù)檢測(cè)鼻咽癌組織中EBER1的轉(zhuǎn)錄表達(dá),篩選鑒定出EBV陽(yáng)性NPC患者。收集EBV陽(yáng)性患者配對(duì)的石蠟包埋組織、咽漱液和PBMC提取DNA,應(yīng)用PCR技術(shù)檢測(cè)EBV1/2分型,PCR結(jié)合限制性片段長(zhǎng)度多態(tài)性(RFLP)分析檢測(cè)F/f分型以及C/D分型,同時(shí)對(duì)EBER和EBNA1基因擴(kuò)增進(jìn)行序列分析。結(jié)果：①20例鼻咽癌組織標(biāo)本中有18例EBER1陽(yáng)性,陽(yáng)性率為90%。②18例陽(yáng)性標(biāo)本中有17例分型成功,1例患者癌組織和咽漱液配對(duì)標(biāo)本1/2分型和F/f分型不同,其余配對(duì)標(biāo)本的EBV基因分型均一致,同時(shí)以1型、F型及C型為主。③在18例配對(duì)標(biāo)本的EBER和EBNA1基因序列分析中,有7例標(biāo)本EBER和EBNA1基因擴(kuò)增成功,其中2例患者癌組織和咽漱液配對(duì)標(biāo)本在EBER基因,1例在EBNA1基因分析中表現(xiàn)為不同的序列變異,其余患者配對(duì)的癌組織和咽漱液中表現(xiàn)為相同序列,即同一患者癌組織和咽漱液是由同一種EBV毒株感染所致。④由于血液標(biāo)本較難擴(kuò)增,同一患者咽漱液、癌組織和PBMC配對(duì)標(biāo)本僅有3例PCR擴(kuò)增成功,其中1例在EBER基因2例在EBNA1基因分析中三種不同部位來(lái)源的EBV基因序列一致。結(jié)論：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由同一種EBV毒株感染。臨床咽漱液標(biāo)本的采集更為方便,提示鼻咽癌患者咽漱液中的EBV分型對(duì)于分析組織中的EBV毒株具有一定的指導(dǎo)意義。但是由于在個(gè)別患者中仍存在不一致的情況,因此在實(shí)驗(yàn)分析過(guò)程中應(yīng)謹(jǐn)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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.63

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