摘要
目的 检测广州地区鼻咽癌组织EB病毒LMP1基因N -末端区XhoI酶切位点的丢失 ,探讨LMP1基因变异在鼻咽癌发生发展中的作用。方法 收集中山大学肿瘤防治中心鼻咽癌新鲜活检标本 63例以及EB病毒健康携带者外周血单个核细胞 10例。采用QIAampDNAMiniKit和QIAampDNABloodMiniKit分别抽取组织和外周血单个核细胞的DNA ,应用巢式PCR扩增EB病毒LMP1基因的N -末端区 ,并用XhoI对扩增产物进行酶切。采用四色荧光末端终止法对扩增产物进行序列分析。结果 10例健康携带者外周血单个核细胞的EB病毒LMP1基因N -末端区均未见XhoI酶切位点的丢失。 63例鼻咽癌组织中有 5 0例 ( 79%)出现XhoI酶切位点的丢失 (XhoI -loss) ,4例 ( 6%)为XhoI酶切位点部分丢失 ,只有 9例 ( 14 %)未见XhoI酶切位点的丢失 (wt -XhoI)。除了XhoI酶切位点的丢失 (nt:16942 3～16942 8;GAGCTC→GATCTC)外 ,还发现 4个错义点突变。结论 广州地区EB病毒健康携带者外周血单个核细胞所携带的EB病毒LMP1基因为wt -XhoI ,而在鼻咽癌组织中主要为XhoI -loss。因此 ,我们认为EB病毒LMP1基因N -末端区XhoI酶切位点的丢失和其他的错义点突变可能是在鼻咽癌的发生发展过程中产生的。
Objective To investigate the loss of an XhoI-site within the N-terminal region of Epstein-Barr virus (EBV) LMP1 gene isolated from nasopharyngeal carcinoma (NPC) for the sake of understanding the sequence variation of EBV LMP1 gene involved in carcinogenesis. Methods Sixty-three fresh nasopharyngeal biopsies taken from patients with NPC were collected from Cancer Center of Sun Yat-sen University. The mononuclear cells of 2-ml perpheral blood (PBMCs) taken from 10 healthy EBV carriers were as a control. The QIAamp DNA Mini Kit and QIAamp DNA Mini Blood Kit were used for extracting the DNA of biopsies and PBMCs, respectively. The N-terminal region of EBV LMP1 was amplified by using nested PCR and then followed by XhoI enzyme digestion. Bidirectional solid-phase sequencing of the PCR product was performed by using the four-coloured fluorescence terminator sequencing method. Results No loss of an XhoI-site within N-terminal region of EBV LMP1 (wt-XhoI) was detected in all 10 carriers' PBMCs. Loss of an XhoI-site (XhoI-loss) was demonstrated in 50 out of 63 NPCs (79 4%) singly and partial loss in 4 of 63 NPCs (6 4%). Single wt-XhoI was found in 9 NPCs (14 3%). Beisdes loss of an XhoI-site (nt:169423～169428; GAGCTC→GATCTC), there were 4 additional missense point mutations found. Conclusion The PBMCs of EBV carriers residing in Guangzhou contain EBV with single wt-XhoI LMP 1 gene, while the XhoI-loss LMP 1 gene of EBV was the predominent variant detected in NPC tissues. There for the genomic alteration within N-terminal region (loss of an XhoI-site and other missense point mutations) of EBV LMP 1 gene may be developed in the process of nasopharyngeal carcinogenesis.
出处
《广东医学》
CAS
CSCD
2003年第11期1170-1172,共3页
Guangdong Medical Journal
基金
国家自然科学基金资助项目 (编号 :39730 2 0 0 -Ⅱ )
