本文選題：EB病毒 + 人皰疹病毒��； 參考：《青島大學》2012年碩士論文

【摘要】：目的EB病毒(Epstein-Barr Virus, EBV)屬皰疹病毒科γ亞科成員,與多種人類腫瘤的發(fā)生密切相關,如Burkit's淋巴瘤(Burkitt's lyrnphoma,BL)、何杰金病(Hodgkin's disease, HD)、鼻咽癌(nasopharyngeal carcinoma, NPC)、乳腺癌以及胃癌(gastric carcinoma, GC)等。有研究表明有超過90%成人感染過EBV,但EBV相關腫瘤的分布則具有明顯的地域性和人群傾向性,除環(huán)境因素和遺傳因素外,是否存在與EBV相關腫瘤有關的EBV基因變異一直是倍受關注的熱點問題。本研究選擇我國山東地區(qū)EBV陽性NPC、 EBV相關胃癌(EBV associated gastric carcinoma, EBVaGC)和健康人群咽漱液(throat washing, TW)作為研究對象,對EBV核抗原3C(EBV nuclear antigen3C, EBNA3C)基因的多態(tài)性進行檢測和分析,旨在探討EBV基因多態(tài)性及其在EBV陽性NPC和EBVaGC發(fā)生中的意義。 方法①聚合酶鏈反應(polymerase chain reaction, PCR)結合DNA測序檢測EBV陽性NPC, EBVaGC, TW標本中EBNA3C基因的多態(tài)性,應用DNAStar5.0軟件中MegAlign對序列進行對比分析,并與EBV標準株B95-8序列進行比較。②根據(jù)測序結果,將其序列標準化處理之后,應用DNAStar5.0軟件生成系統(tǒng)樹。③根據(jù)各基因出現(xiàn)的共有突變結合系統(tǒng)樹對各基因變異進行分類,初步分析基因變異對相關基因功能的影響。④比較NPC、 EBVaGC和健康人群EBNA3C基因分型及基因變異各類型的分布,并與以往文獻發(fā)表的相關數(shù)據(jù)進行比較和分析,以探尋具有中國北方特異性的變異。 結果103例標本成功進行PCR擴增及測序,包括26例EBVaGC,50例NPC及27例健康成人咽漱液,根據(jù)測序結果及系統(tǒng)樹,所有標本又可以分為三個亞型,分別為3C-5,3C-6,3C-12。 ①3C-6亞型為主要亞型,見于17例EBVaGCs(17/26,65.4%),25例NPC(25/50,50(?)%)以及14例TW(14/27,51.9%)。此亞型共發(fā)現(xiàn)有6個共有突變,分別為氨基酸655(Gly→Ser),677(Thr→Met),683(Ala→Val),687(Cys→Thr),和246(gta→gtg)。 ②3C-12亞型為次要亞型,見于8例EBVaGCs(8/2630.8%),11例NPC(11/5022.0%)以及6例TW(6/2722.2%)。共鑒定出12個共有突變,分別為AA208(gcc→gct),213(Gln→His),336(Glu→Asp),337(cac→cat),AA348(Ile→Leu), AA367(gaa→gag),370(agt→agc),414(aag→aaa),467(ccg→cct),656(Arg→Gly),687(Cys→Thr),701(Glu→Gln). ③3C-5共有5個共有突變,分別為AA348(Ile→Leu), AA367(gaa→gag),370(agt→agc),687(Cys→Thr),701(Glu→Gln)。此亞型見于1例EBVaGC1/263.8%),14例NPC(14/5028.0%)以及7例TW(7/2725.9%)。 ④抗原表位突變。EBNA3C8個抗原表位中共有4個發(fā)生表位突變,分別為QNG (213Gln→His), LRG (254Gln→His), KEH (336Glu→Asp)和FRK(348Ile→Leu),其他表位高度保守。 ⑤統(tǒng)計學分析結果顯示,EBVaGC, NPC及TW中EBNA3C各基因亞型的分布無顯著性差異(P=0.17) 結論 ①3C-6亞型是中國山東地區(qū)主要亞型,見于56例標本中(54.4%,56/103)。變異類型與中國南方和日本地區(qū)基本相同。 ②EBNA3C各亞型在EBVaGC、 NPC和健康人咽漱液中的分布無明顯統(tǒng)計學差異,提示EBNA3C基因多態(tài)性具有地域特點而非腫瘤特異。 ③首次觀察到3C-5(21.4%,22/103)這一新的亞型,該亞型之前未見報道,有可能為中國北方地區(qū)的特異型別。 ④我們所測的EBNA3C8個抗原表位中共有4處出現(xiàn)變異,這些變異可能會影響內(nèi)源性抗原處理、主要組織相容性復合體(MHC)結合及T細胞受體對抗原肽-MHC的識別,進而導致病毒在宿主體內(nèi)長存。
[Abstract]:Objective EB virus (Epstein-Barr Virus, EBV) is a member of the family of herpes simplex family, which is closely related to the occurrence of a variety of human tumors, such as Burkit's lymphoma (Burkitt's lyrnphoma, BL), Hodgkin's disease (Hodgkin's disease, HD), nasopharyngeal carcinoma (nasopharyngeal), breast and gastric cancer. More than 90% adults were infected with EBV, but the distribution of EBV related tumors was marked regionalism and population tendency. In addition to environmental and genetic factors, the existence of EBV gene mutations associated with EBV related tumors has always been a hot issue. This study chose EBV positive NPC in Shandong and EBV related gastric cancer (EBV). Associated gastric carcinoma, EBVaGC) and healthy population of throat washing (TW) were used as research objects to detect and analyze the polymorphism of EBV nuclear antigen 3C (EBV nuclear antigen3C) gene, aiming at exploring the genetic polymorphism and its significance in the occurrence and occurrence of EBV.
Methods polymerase chain reaction (PCR) and DNA sequencing were used to detect the polymorphisms of EBNA3C genes in EBV positive NPC, EBVaGC, and TW specimens. The sequences were compared with MegAlign pairs in DNAStar5.0 software and compared with those of the standard sequence. 2. After sequencing the sequence, the sequence was standardized. DNAStar5.0 software was used to generate the system tree. Thirdly, according to the common mutation and system tree of each gene, the variation of gene mutation was classified, and the effect of gene mutation on the function of related genes was preliminarily analyzed. (4) the distribution of NPC, EBVaGC and EBNA3C genotyping and gene variation in healthy people were compared and related to the previous literature. Data were compared and analyzed to explore specific variability in northern China.
Results 103 cases were successfully amplified and sequenced by PCR, including 26 cases of EBVaGC, 50 cases of NPC and 27 healthy adult pharynx gargle. According to the sequencing results and the system tree, all the specimens could be divided into three subtypes, respectively, 3C-5,3C-6,3C-12..
(1) 3C-6 subtypes were the main subtypes in 17 cases of EBVaGCs (17/26,65.4%), 25 cases of NPC (25/50,50 (?)%) and 14 cases of TW (14/27,51.9%). This subtype found a total of 6 common mutations, which were amino acids 655 (Gly to Ser), 677 (Thr to Met), 683 (Ala to), 687, 246, and 246.
(2) 3C-12 subtypes were secondary subtypes in 8 cases of EBVaGCs (8/2630.8%), 11 cases of NPC (11/5022.0%) and 6 cases of TW (6/2722.2%). There were 12 common mutations, respectively AA208 (GCC > GCT), 213 (Gln > His), 336, 337, 370, 467, 467, 656. 687 (Cys to Thr), 701 (Glu to Gln).
(3) there are 5 common mutations in 3C-5, AA348 (Ile to Leu), AA367 (GAA to GAG), 370 (AGT to AGC), 687 (Cys to Thr), 701 (Glu to). This subtype is seen in 1 cases), 14 cases and 7 cases.
(4) there are 4 epitopes in the epitopes of.EBNA3C8 antigen epitopes, which are QNG (213Gln to His), LRG (254Gln to His), KEH (336Glu to Asp) and FRK (348Ile to), and the other epitopes are highly conservative.
Statistical analysis showed that there was no significant difference in the distribution of EBNA3C gene subtypes in EBVaGC, NPC and TW (P=0.17).
conclusion
(1) the 3C-6 subtype is a major subtype in Shandong, China. It is found in 56 specimens (54.4%, 56/103). The variation type is basically the same as that in southern China and Japan.
There was no significant difference in the distribution of EBNA3C subtypes in EBVaGC, NPC and healthy people's pharynx gargle, suggesting that the polymorphism of EBNA3C gene has regional characteristics, not tumor specific.
(3) a new subtype of 3C-5 (21.4%, 22/103) has been observed for the first time. This subtype has not been reported before, and may be a specific type in northern China.
(4) there are 4 mutations in the EBNA3C8 epitopes we have measured, which may affect endogenous antigen processing, the binding of the main histocompatibility complex (MHC) and the recognition of the T cell receptor against the propeptide -MHC, which leads to the survival of the virus in the host.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R373

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