摘要
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 sequence variations frequently found within the N- and C- terminal regions of Epstein-Barr virus （EBV） LMP1 gene in nasopharyngeal carcinoma （NPC） and to study the underlying mechanisms. Methods Fresh tumor tissues were sampled from 63 patients with untreated NPC encountered in Affiliated Tumor Hospital of Sun Yat-sen University, Guangzhou. The N-terminal region of EBV LMP1 gene was amplified with nested polymerase chain reaction （ PCR）, followed by XhoⅠ enzyme digestion. Nested PCR was also employed to detect the 30 base pairs deletion within the C-terminal region. Four-colored fluorescence terminator sequencing method was applied for hi-directional solid-phase sequencing of the 8 representative PCR products in 4 cases of NPC. The DNA sequence within the N- and C-terminal regions of LMP1 gene was then analyzed. Results There were 4 patterns of sequence variations, namely, wt-XhoⅠ/wt-LMP1 （4 cases, 6. 3% ） , wt-XhoⅠ and XhoⅠ -loss/del-LMP1 （4 cases, 6. 3% ）, wt-XhoⅠ/del-LMP1 （5 cases, 7.9% ） and XhoⅠ-loss/del-LMP1 （50 cases, 79.5% ）, detected in the 63 studied cases. Sequence analysis showed that the EBV LMP1 gene had underwent non-synonymous and synonymous substitutions, as compared with the prototype of B95-8 cells. The ratio of non-synonymous to synonymous substitutions was 2. 25. Conclusions XhoⅠ-loss/del-LMP1 is the predominant sequence variation pattern of EBV LMP1 gene in NPC from Guangzhou. The XhoⅠ -loss variation seems to develop on top of del-LMP1. When compared with the EBV LMP1 gene in peripheral blood B-lymphocytes of virus carriers and in preinvasive epithelial lesions （reported previously）, it is likely that the sequence variation patterns of LMP1 gene may represent 4 different phases of intrahost evolution of EBV during nasopharyngeal carcinogenesis.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2005年第12期791-795,共5页
Chinese Journal of Pathology
基金
国家自然科学基金资助项目(39730200-Ⅱ)
