山东地区EBV核抗原3C基因多态性的研究

发布时间：2018-06-19 07:43

本文选题：EB病毒 + 人疱疹病毒　；参考：《青岛大学》2012年硕士论文

【摘要】：目的EB病毒(Epstein-Barr Virus, EBV)属疱疹病毒科γ亚科成员,与多种人类肿瘤的发生密切相关,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。有研究表明有超过90%成人感染过EBV,但EBV相关肿瘤的分布则具有明显的地域性和人群倾向性,除环境因素和遗传因素外,是否存在与EBV相关肿瘤有关的EBV基因变异一直是倍受关注的热点问题。本研究选择我国山东地区EBV阳性NPC、 EBV相关胃癌(EBV associated gastric carcinoma, EBVaGC)和健康人群咽漱液(throat washing, TW)作为研究对象,对EBV核抗原3C(EBV nuclear antigen3C, EBNA3C)基因的多态性进行检测和分析,旨在探讨EBV基因多态性及其在EBV阳性NPC和EBVaGC发生中的意义。方法①聚合酶链反应(polymerase chain reaction, PCR)结合DNA测序检测EBV阳性NPC, EBVaGC, TW标本中EBNA3C基因的多态性,应用DNAStar5.0软件中MegAlign对序列进行对比分析,并与EBV标准株B95-8序列进行比较。②根据测序结果,将其序列标准化处理之后,应用DNAStar5.0软件生成系统树。③根据各基因出现的共有突变结合系统树对各基因变异进行分类,初步分析基因变异对相关基因功能的影响。④比较NPC、 EBVaGC和健康人群EBNA3C基因分型及基因变异各类型的分布,并与以往文献发表的相关数据进行比较和分析,以探寻具有中国北方特异性的变异。结果103例标本成功进行PCR扩增及测序,包括26例EBVaGC,50例NPC及27例健康成人咽漱液,根据测序结果及系统树,所有标本又可以分为三个亚型,分别为3C-5,3C-6,3C-12。 ①3C-6亚型为主要亚型,见于17例EBVaGCs(17/26,65.4%),25例NPC(25/50,50(?)%)以及14例TW(14/27,51.9%)。此亚型共发现有6个共有突变,分别为氨基酸655(Gly→Ser),677(Thr→Met),683(Ala→Val),687(Cys→Thr),和246(gta→gtg)。 ②3C-12亚型为次要亚型,见于8例EBVaGCs(8/2630.8%),11例NPC(11/5022.0%)以及6例TW(6/2722.2%)。共鉴定出12个共有突变,分别为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个共有突变,分别为AA348(Ile→Leu), AA367(gaa→gag),370(agt→agc),687(Cys→Thr),701(Glu→Gln)。此亚型见于1例EBVaGC1/263.8%),14例NPC(14/5028.0%)以及7例TW(7/2725.9%)。 ④抗原表位突变。EBNA3C8个抗原表位中共有4个发生表位突变,分别为QNG (213Gln→His), LRG (254Gln→His), KEH (336Glu→Asp)和FRK(348Ile→Leu),其他表位高度保守。 ⑤统计学分析结果显示,EBVaGC, NPC及TW中EBNA3C各基因亚型的分布无显著性差异(P=0.17) 结论 ①3C-6亚型是中国山东地区主要亚型,见于56例标本中(54.4%,56/103)。变异类型与中国南方和日本地区基本相同。 ②EBNA3C各亚型在EBVaGC、 NPC和健康人咽漱液中的分布无明显统计学差异,提示EBNA3C基因多态性具有地域特点而非肿瘤特异。 ③首次观察到3C-5(21.4%,22/103)这一新的亚型,该亚型之前未见报道,有可能为中国北方地区的特异型别。 ④我们所测的EBNA3C8个抗原表位中共有4处出现变异,这些变异可能会影响内源性抗原处理、主要组织相容性复合体(MHC)结合及T细胞受体对抗原肽-MHC的识别,进而导致病毒在宿主体内长存。
[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.
【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】：R373

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