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目的检测广州地区鼻咽癌中EB病毒LMP1基因N-和C-末端区序列变异的热点,并探讨其产生的机制。方法收集中山大学肿瘤医院未经治疗的鼻咽癌患者鼻咽新鲜活检标本63例。采用巢式聚合酶链反应(PCR)扩增EB病毒LMP1基因N-和C-末端区,用XhoⅠ对N-末端区扩增产物进行酶切分析,并检测C-末端区扩增产物30bp缺失的情况。采用四色荧光末端终止法对4例患者N-和C-末端区的8份扩增产物进行序列分析。结果63例鼻咽癌组织EB病毒LMP1基因N-和C-末端区有4种序列变异类型:wt-XhoⅠ/wt-LMP1(4例,6·3%)、wt-XhoⅠ&XhoⅠ-loss/del-LMP1(4例,6·3%)、wt-XhoⅠ/del-LMP1(5例,7·9%)和XhoⅠ-loss/del-LMP1(50例,79·5%)。序列分析显示:与B95-8细胞相比较,所有检测的鼻咽癌组织中EB病毒LMP1基因均发生了错义点突变和无义点突变。错义点突变数与无义点突变数之间的比值为2·25(9/4)。结论广州地区鼻咽癌中EB病毒LMP1基因的序列变异类型以XhoⅠ-loss/del-LMP1占主导。LMP1基因N-末端区XhoⅠ酶切位点的丢失很可能是在C-末端区30bp缺失的基础上发生的。LMP1基因的4种序列变异类型可能代表了鼻咽癌变过程中EB病毒在宿主内演变的4个时相。
Objective To detect the hot spots in the sequence variation of N-terminal and C-terminal regions of Epstein-Barr virus (Epstein-Barr virus) in nasopharyngeal carcinoma in Guangzhou and to explore its mechanism. Methods Totally 63 nasopharyngeal fresh biopsy specimens from patients with nasopharyngeal carcinoma untreated in Sun Yat-sen University Cancer Hospital were collected. N-and C-terminal regions of EBV LMP1 gene were amplified by nested polymerase chain reaction (PCR) and digested with Xho I to amplify the N-terminal region. The 30 bp deletion of the C-terminal region was detected Case. Eight amplified products from the N- and C-terminal regions of four patients were sequenced using the four-color fluorescent end-termination method. Results There were 4 types of sequence variation in the N- and C-terminal regions of EBV LMP1 gene in 63 cases of nasopharyngeal carcinoma: wt-XhoⅠ / wt-LMP1 (4 cases, 6.3%), wt-XhoⅠ & XhoⅠ-loss / LMP1 (4 cases, 6.3%), wt-XhoI / del-LMP1 (5 cases, 7.9%) and XhoI-loss / del-LMP1 (50 cases, 79.5%). Sequence analysis showed that compared with B95-8 cells, all the detected NPC virus LMP1 gene mutations have missense point mutations and nonsense point mutations. The ratio of missense point mutations to nonsense point mutations was 2.25 (9/4). Conclusions The sequence variation of LMP1 gene in nasopharyngeal carcinoma in Guangzhou is dominated by XhoⅠ-loss / del-LMP1. The loss of the XhoI restriction site at the N-terminal region of the LMP1 gene is likely to occur on the basis of a 30 bp deletion in the C-terminal region. Four types of sequence variation of LMP1 gene may represent four phases of Epstein-Barr virus evolution in the host during nasopharyngeal carcinogenesis.