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[摘要] 目的 探討EB病毒(EBV)编码基因变异与血液肿瘤的关系。
方法选取山东地区白血病病人186例和骨髓增生异常综合征(MDS)病人77例作为研究对象,采用巢式PCR技术分析EBV 1/2分型,PCR结合限制性酶切技术分析EBV F/f分型,巢式PCR和DNA测序检测EBV编码小RNA(EBERs,包括EBER1和EBER2)基因的变异情况。
结果白血病和MDS病人1型EBV的检出率分别为77.3%(51/66)和89.3%(25/28),2型EBV检出率分别为7.6%(5/66)和7.1%(2/28),同时携带1型和2型两种基因型检出率分别为15.1%(10/66)和3.6%(1/28);F型EBV在白血病和MDS中的检出率分别为98.6%(68/69)和91.7%(22/24),f型EBV在白血病和MDS中的检出率分别为1.4%(1/69)和8.3%(2/24)。白血病和MDS中1/2分型、F/f分型的分布比较,差异无显著性(P>0.05)。白血病和MDS中EBERs的主要基因型为EB-6m型,分别为85.0%(34/40)和75.0%(15/20),差异无显著性(P>0.05)。
结论山东地区白血病和MDS的EBV基因型以1型和F型为主,EBERs的优势基因型为EB-6m,EB-6m型EBERs可能与白血病发生相关。
[关键词] 疱疹病毒4型,人;白血病;骨髓增生异常综合征;多态性,单核苷酸
[中图分类号] R733.7
[文献标志码] A
[文章编号] 2096-5532(2019)01-0050-05
AN ANALYSIS OF EPSTEIN-BARR VIRUS-ENCODED GENE VARIATIONS IN PATIENTS WITH HEMATOLOGIC MALIGNANCIES IN SHANDONG, CHINA
WANG Haiyu, SUN Lingling, LI Ping, LIU Wen, ZHANG Zhongguang
(Department of Medical Microbiology, Qingdao University Medical College, Qingdao 266021, China)
[ABSTRACT]ObjectiveTo investigate the association between hematologic malignancies and the genotypes of Epstein-Barr virus (EBV)-encoded genes.
MethodsA total of 186 patients with leukemia and 77 patients with myelodysplastic syndrome (MDS) in Shandong, China were selected as subjects. EBV 1/2 type was analyzed by nested polymerase chain reaction (PCR), while EBV F/f type was analyzed by PCR combined with restriction fragment length polymorphism. And the genotypes of EBV-encoded small RNAs (EBERs, including EBER1 and EBER2) were analyzed by nested PCR and DNA sequencing.
Results
In the patients with leukemia and MDS, the detection rates of EBV type 1 were 77.3% (51/66) and 89.3% (25/28), respectively; the detection rates of EBV type 2 were 7.6% (5/66) and 7.1% (2/28), respectively; the detection rates of both EBV type 1 and 2 were 15.1% (10/66) and 3.6% (1/28), respectively; the detection rates of EBV type F were 98.6% (68/69) and 91.7% (22/24), respectively; the detection rates of EBV type f were 1.4% (1/69) and 8.3% (2/24), respectively. There were no significant differences in the distributions of EBV type 1/2 and EBV type F/f between leukemia and MDS (P>0.05). The dominant genotype of EBERs was EB-6m, and there was no significant difference in the distribution of EB-6m between leukemia and MDS (85.0% (34/40) vs 75.0% (15/20), P>0.05).
ConclusionType 1 and type F are the main EBV genotypes in patients with leukemia and those with MDS in Shandong. The dominant genotype of EBERs is EB-6m, which may be associated with leukemia. [KEY WORDS]herpesvirus 4, human; leukemi; myelodysplastic syndromes; polymorphism, single nucleotide
EB病毒(EBV)是重要的DNA肿瘤病毒,与鼻咽癌、胃癌和霍奇金淋巴瘤等多种肿瘤的发生发展密切相关[1-5]。近年来,有学者对EBV感染与白血病的关系进行了研究,结果显示EBV独特的表达模式是B细胞特殊分化窗口的标志,可导致慢性白血病[6]。ATEYAH等[7]研究发现,感染EBV的B细胞急性淋巴细胞白血病病人的CD4、CD25High+、Foxp3+、Treg因子的表达明显增加。GUAN等[8]研究发现,EBV感染与急性白血病的发生有关。然而,关于EBV在血液肿瘤中的潜在作用目前仍不清楚。EBV基因型存在变异,有关EBV基因多态性的研究已经发现了多种EBV亚型,但EBV编码基因变异与肿瘤发生发展的关系仍没有明确。根据EBV核抗原2和3的序列差异(分别为EBNA-2和
EBNA-3)可以将EBV分为基因型1和2(又称a和b);根据BamHⅠ F片段缺乏或出现BamHⅠ识别位点,采用PCR结合限制性片段长度多态性(PCR-RFLP)分析技术可以区分F/f变异[9-12]。EBV编码小RNA(EBERs)是所有EBV潜伏类型表达最为丰富的mRNA,在每个细胞中的拷贝数为106~107拷贝,因此EBERs已经成为EBV潜伏感染的标志物[13-14]。以往有研究表明,某些EBV基因变异型可能与某些肿瘤的发病机制有关[12,15],如f型EBV和EB-8m型EBERs被认为与鼻咽癌尤其是高发区鼻咽癌相关[16-17]。我们曾对山东地区胃癌、鼻咽癌、淋巴瘤及健康人群中多个EBV编码基因的多态性进行了研究[16-18],但尚不清楚EBV编码基因在恶性血液病中的变异情况及其生物学意义。本研究检测白血病和骨髓增生异常综合征(MDS)病人中EBV 1/2分型、F/f分型以及EBERs基因多态性,探讨其与血液肿瘤的关系。
1 材料与方法
1.1 标本采集
2017年6月—2018年1月,收集青岛大学附属医院住院及门诊白血病病人186例,其中急性白血病(AL)病人125例,慢性白血病(CL)病人61例;MDS病人77例。取病人外周血标本4 mL, EDTA抗凝,1 200 r/min离心10 min,吸取中间白细胞层,采用蛋白酶K消化法和酚氯仿法提取细胞DNA。
1.2EBV基因型1/2和F/f的分型方法
采用巢式PCR方法,根据EBNA-2编码序列差异,对EBV基因型1/2进行分析[19]。根据BamHⅠF片段缺乏或出现BamHⅠ识别位点,采用PCR-RFLP技术分析EBV分离毒株F/f基因型[20]。PCR引物序列和相应扩增产物大小见表1。
1.3 EBERs基因扩增
采用巢式PCR扩增EBERs基因(nt6629~nt7128),外侧PCR反应体系为10×Buffer 2.0 μL,dNTPs浓度0.2 mmol/L,高保真Taq DNA聚合酶1.0 U,上下游引物各0.4 μmol/L,以及DNA模板100 ng。循环条件为:94 ℃预变性5 min;然后94 ℃变性30 s,55 ℃退火30 s,72 ℃延伸2 min,共扩增35个循环;最后72 ℃延伸10 min。取外侧引物PCR产物2 μL为内侧引物PCR的模板,其反应体系和循环条件同上。PCR产物经12 g/L琼脂糖凝胶电泳分析。阳性产物送北京华大基因公司进行全长测序,测序后将序列与EBV原型B95-8(GenBank:v01555.2)进行比较。
1.4 统计学分析
采用SPSS 15.0统计软件(SPSS,Chicago,IL)进行分析,数据间比较采用 χ2检验及精确概率法。以P<0.05为差异有统计学意义。
2 结 果
2.1 白血病和MDS病人EBV检出情况
本文186例白血病标本中有69例(37.1%)、77例MDS中有28例(36.4%)检测到EBV DNA,两组间EBV检出率比较差异无显著性(P>0.05)。
2.2 EBV基因分型
对检测到EBV DNA标本进行扩增,69例白血病标本3例扩增失败,另外66例样本中有51例
(77.3%)、28例MDS样本中有25例(89.3%)检出1型EBV;66例白血病标本中5例(7.6%)、28例MDS标本中2例(7.1%)检出2型EBV。其中10例白血病标本(15.1%)和1例MDS(3.6%)同时携带1型和2型两种基因型。见表2。本文69例EBV阳性白血病标本分别有68例(98.6%)和1例(1.8%)为F型和f型,24例MDS标本中基因型F和f分别为22例(91.7%)和2例(8.3%)。见表2。表2中同时列出了相关文献检测到的同一地区健康人群中EBV 1/2分型和F/f分型的结果[16,18]。白血病、MDS以及健康人群中1/2分型的分布差异无显著性(P>0.05),F/f分型的分布差异亦无显著性(χ2=3.030,P>0.05)。
2.3 EBERs基因的序列变异
本文40例白血病和20例MDS标本EBERs基因相应片段扩增成功,其变异情况见图1。根据相关文献分类方法[18],检测到两种EBERs基因型:EB-6m和B95-8。11例标本为B95-8型序列,包括6例(15.0%)白血病标本和5例(25.0%)MDS标本,B95-8型序列与EBV标准株序列相同或仅出现散在无规律突变,见图1。49例标本为EB-6m型,包括34例(85.0%)白血病和15例(75.0%)MDS标本,见表2。与B95-8比较,EB-6m具有6处共有突变,除1处(7123nt A→G)位于EBER2基因,其余5处均位于EBER1和EBER2基因之间161 bp(nt6796~6955)间隔序列中,分别为6808nt T→A、6884nt G→A、6886nt T→G、6911nt A→G和6944nt G→A。其中1例MDS标本中EB-6m亚型只有5个共有突变序列(6808 nt、6884 nt、6911 nt、6944 nt和7123 nt)。除共有突变外,部分样本还出现其他位点的散在突变(图1)。EBERs变异类型在白血病和MDS中分布差异无显著性(P>0.05),但白血病、MDS與以往同一地区健康人群之间的EBERs基因变异类型的分布不同,差异有统计学意义(χ2=5.455、8.706,P<0.01)。 圖中第2行数字对应于核苷酸位点,B95-8核苷酸序列在第2行下面。第1列为标本类型,第2列为不同的基因亚型;图中第3列把相同序列的标本合并,以1例标本为其代表株,标本编号后括号内数字为该相同序列的标本数。图中大写字母表示核苷酸。
3 讨 论
EBV可能在白血病和MDS发病中发挥着重要的作用[21-23]。本文的研究结果显示,186例白血病样本中有69例(37.1%)、77例MDS样本中有28例(36.4%)为EBV阳性标本。白血病和MDS病人中EBV检出率高于健康对照组(5.4%,2/37)[8]。
本研究结果显示,白血病和MDS中1型EBV检出率分别为77.3%(51/66)和89.3%(25/28),F型EBV在白血病和MDS中的检出率分别为98.6%(68/69)和91.7%(22/24),提示山东地区白血病和MDS均以1型和F型EBV为主,这与以往该地区鼻咽癌、胃癌、淋巴瘤和健康人群的研究结果一致,也与世界其他多数地域的研究结果一致[17-18]。以往研究显示,1型EBV是大多数地区的优势毒株,而2型EBV仅在中非和新几内亚占主导地位[24-25];F型EBV是除我国南方地区鼻咽癌高发区以外地域的主要基因型,f型EBV仅在我国南方鼻咽癌高发区较为多见,而且其在高发区鼻咽癌中的检出率高于非鼻咽癌人群,被认为与高发区鼻咽癌相关[18]。
以往研究发现了4种EBERs基因型:EB-6m、EB-8m、EB-10m和B95-8,山东地区EBV相关胃癌、鼻咽癌、淋巴瘤和健康人群以及广东地区健康人群均以EB-6m变异型为主,而EB-8m变异型仅为中国南方鼻咽癌的主要变异型,且其检出率高于健康人群,可能与鼻咽癌尤其是高发区鼻咽癌发生相关[16-18]。已有研究显示,美国的16例霍奇金病有13例(81.3%)EBERs基因型为EB-6m[26],3例EBERs基因型为B95-8;24例移植后淋巴增殖性疾病有19例(79.2%)EBERs基因型为EB-6m[27],5例EBERs基因型为B95-8;德国的41例良性和恶性疾病中有31例(75.6%)EBERs基因型为EB-6m,10例EBERs基因型为B95-8[28];意大利的15例人类免疫缺陷病毒感染的病人有14例(93.3%)EBERs基因型为EB-6m,1例EBERs基因型为B95-8,11例健康人群有7例(63.6%)EBERs基因型为EB-6m,4例EBERs基因型为B95-8[29];南非21例鼻咽癌有18例(85.7%)EBERs基因型为EB-6m,3例EBERs基因型为B95-8[30]。由上述数据可见,EBERs变异除与地理分布有关外,可能还具有疾病关联性。
本研究结果显示,EBERs基因EB-6m亚型是白血病和MDS中最常见的类型,而未发现EB-8m和EB-10 m亚型;白血病和MDS中EBERs基因型EB-6m检出率分别为85.0%(34/40)和75.0%(15/20),低于健康人群(96.7%,89/92),本文结果与以往其他人群中的研究结果相一致[16-17]。因此,EBERs变异在白血病和MDS发病中的作用可能与其他EBV相关肿瘤相同,EB-6m型EBERs可能与白血病及MDS的发生相关,但这需要来自更多地域人群的相关研究加以证实。EBERs是非编码RNA,EBERs可通过促进细胞生长和调节免疫功能而参与鼻咽癌和Burkitt淋巴瘤等肿瘤的发生发展[31]。同时,EBV编码的小RNA(EBERs)可以诱导自分泌生长因子的表达,维持Burkitt淋巴瘤细胞的恶性表型,表明EBERs在肿瘤发生中有潜在作用[32]。近年来,EBV阳性淋巴瘤、胃癌及鼻咽癌细胞系中EBERs在细胞转化中的作用已经被证实[31]。白血病和MDS的发生可能与EBERs在参与免疫调节中某些特异蛋白的表达有关。因此,有必要对EB-6m变异型EBERs及含EB-6m变异型EBERs的EBV毒株进行功能学研究,探讨该变异对致癌潜能以及免疫调节的影响,这对最终阐明EBERs基因变异在白血病和MDS发生中的作用具有重要意义。
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方法选取山东地区白血病病人186例和骨髓增生异常综合征(MDS)病人77例作为研究对象,采用巢式PCR技术分析EBV 1/2分型,PCR结合限制性酶切技术分析EBV F/f分型,巢式PCR和DNA测序检测EBV编码小RNA(EBERs,包括EBER1和EBER2)基因的变异情况。
结果白血病和MDS病人1型EBV的检出率分别为77.3%(51/66)和89.3%(25/28),2型EBV检出率分别为7.6%(5/66)和7.1%(2/28),同时携带1型和2型两种基因型检出率分别为15.1%(10/66)和3.6%(1/28);F型EBV在白血病和MDS中的检出率分别为98.6%(68/69)和91.7%(22/24),f型EBV在白血病和MDS中的检出率分别为1.4%(1/69)和8.3%(2/24)。白血病和MDS中1/2分型、F/f分型的分布比较,差异无显著性(P>0.05)。白血病和MDS中EBERs的主要基因型为EB-6m型,分别为85.0%(34/40)和75.0%(15/20),差异无显著性(P>0.05)。
结论山东地区白血病和MDS的EBV基因型以1型和F型为主,EBERs的优势基因型为EB-6m,EB-6m型EBERs可能与白血病发生相关。
[关键词] 疱疹病毒4型,人;白血病;骨髓增生异常综合征;多态性,单核苷酸
[中图分类号] R733.7
[文献标志码] A
[文章编号] 2096-5532(2019)01-0050-05
AN ANALYSIS OF EPSTEIN-BARR VIRUS-ENCODED GENE VARIATIONS IN PATIENTS WITH HEMATOLOGIC MALIGNANCIES IN SHANDONG, CHINA
WANG Haiyu, SUN Lingling, LI Ping, LIU Wen, ZHANG Zhongguang
(Department of Medical Microbiology, Qingdao University Medical College, Qingdao 266021, China)
[ABSTRACT]ObjectiveTo investigate the association between hematologic malignancies and the genotypes of Epstein-Barr virus (EBV)-encoded genes.
MethodsA total of 186 patients with leukemia and 77 patients with myelodysplastic syndrome (MDS) in Shandong, China were selected as subjects. EBV 1/2 type was analyzed by nested polymerase chain reaction (PCR), while EBV F/f type was analyzed by PCR combined with restriction fragment length polymorphism. And the genotypes of EBV-encoded small RNAs (EBERs, including EBER1 and EBER2) were analyzed by nested PCR and DNA sequencing.
Results
In the patients with leukemia and MDS, the detection rates of EBV type 1 were 77.3% (51/66) and 89.3% (25/28), respectively; the detection rates of EBV type 2 were 7.6% (5/66) and 7.1% (2/28), respectively; the detection rates of both EBV type 1 and 2 were 15.1% (10/66) and 3.6% (1/28), respectively; the detection rates of EBV type F were 98.6% (68/69) and 91.7% (22/24), respectively; the detection rates of EBV type f were 1.4% (1/69) and 8.3% (2/24), respectively. There were no significant differences in the distributions of EBV type 1/2 and EBV type F/f between leukemia and MDS (P>0.05). The dominant genotype of EBERs was EB-6m, and there was no significant difference in the distribution of EB-6m between leukemia and MDS (85.0% (34/40) vs 75.0% (15/20), P>0.05).
ConclusionType 1 and type F are the main EBV genotypes in patients with leukemia and those with MDS in Shandong. The dominant genotype of EBERs is EB-6m, which may be associated with leukemia. [KEY WORDS]herpesvirus 4, human; leukemi; myelodysplastic syndromes; polymorphism, single nucleotide
EB病毒(EBV)是重要的DNA肿瘤病毒,与鼻咽癌、胃癌和霍奇金淋巴瘤等多种肿瘤的发生发展密切相关[1-5]。近年来,有学者对EBV感染与白血病的关系进行了研究,结果显示EBV独特的表达模式是B细胞特殊分化窗口的标志,可导致慢性白血病[6]。ATEYAH等[7]研究发现,感染EBV的B细胞急性淋巴细胞白血病病人的CD4、CD25High+、Foxp3+、Treg因子的表达明显增加。GUAN等[8]研究发现,EBV感染与急性白血病的发生有关。然而,关于EBV在血液肿瘤中的潜在作用目前仍不清楚。EBV基因型存在变异,有关EBV基因多态性的研究已经发现了多种EBV亚型,但EBV编码基因变异与肿瘤发生发展的关系仍没有明确。根据EBV核抗原2和3的序列差异(分别为EBNA-2和
EBNA-3)可以将EBV分为基因型1和2(又称a和b);根据BamHⅠ F片段缺乏或出现BamHⅠ识别位点,采用PCR结合限制性片段长度多态性(PCR-RFLP)分析技术可以区分F/f变异[9-12]。EBV编码小RNA(EBERs)是所有EBV潜伏类型表达最为丰富的mRNA,在每个细胞中的拷贝数为106~107拷贝,因此EBERs已经成为EBV潜伏感染的标志物[13-14]。以往有研究表明,某些EBV基因变异型可能与某些肿瘤的发病机制有关[12,15],如f型EBV和EB-8m型EBERs被认为与鼻咽癌尤其是高发区鼻咽癌相关[16-17]。我们曾对山东地区胃癌、鼻咽癌、淋巴瘤及健康人群中多个EBV编码基因的多态性进行了研究[16-18],但尚不清楚EBV编码基因在恶性血液病中的变异情况及其生物学意义。本研究检测白血病和骨髓增生异常综合征(MDS)病人中EBV 1/2分型、F/f分型以及EBERs基因多态性,探讨其与血液肿瘤的关系。
1 材料与方法
1.1 标本采集
2017年6月—2018年1月,收集青岛大学附属医院住院及门诊白血病病人186例,其中急性白血病(AL)病人125例,慢性白血病(CL)病人61例;MDS病人77例。取病人外周血标本4 mL, EDTA抗凝,1 200 r/min离心10 min,吸取中间白细胞层,采用蛋白酶K消化法和酚氯仿法提取细胞DNA。
1.2EBV基因型1/2和F/f的分型方法
采用巢式PCR方法,根据EBNA-2编码序列差异,对EBV基因型1/2进行分析[19]。根据BamHⅠF片段缺乏或出现BamHⅠ识别位点,采用PCR-RFLP技术分析EBV分离毒株F/f基因型[20]。PCR引物序列和相应扩增产物大小见表1。
1.3 EBERs基因扩增
采用巢式PCR扩增EBERs基因(nt6629~nt7128),外侧PCR反应体系为10×Buffer 2.0 μL,dNTPs浓度0.2 mmol/L,高保真Taq DNA聚合酶1.0 U,上下游引物各0.4 μmol/L,以及DNA模板100 ng。循环条件为:94 ℃预变性5 min;然后94 ℃变性30 s,55 ℃退火30 s,72 ℃延伸2 min,共扩增35个循环;最后72 ℃延伸10 min。取外侧引物PCR产物2 μL为内侧引物PCR的模板,其反应体系和循环条件同上。PCR产物经12 g/L琼脂糖凝胶电泳分析。阳性产物送北京华大基因公司进行全长测序,测序后将序列与EBV原型B95-8(GenBank:v01555.2)进行比较。
1.4 统计学分析
采用SPSS 15.0统计软件(SPSS,Chicago,IL)进行分析,数据间比较采用 χ2检验及精确概率法。以P<0.05为差异有统计学意义。
2 结 果
2.1 白血病和MDS病人EBV检出情况
本文186例白血病标本中有69例(37.1%)、77例MDS中有28例(36.4%)检测到EBV DNA,两组间EBV检出率比较差异无显著性(P>0.05)。
2.2 EBV基因分型
对检测到EBV DNA标本进行扩增,69例白血病标本3例扩增失败,另外66例样本中有51例
(77.3%)、28例MDS样本中有25例(89.3%)检出1型EBV;66例白血病标本中5例(7.6%)、28例MDS标本中2例(7.1%)检出2型EBV。其中10例白血病标本(15.1%)和1例MDS(3.6%)同时携带1型和2型两种基因型。见表2。本文69例EBV阳性白血病标本分别有68例(98.6%)和1例(1.8%)为F型和f型,24例MDS标本中基因型F和f分别为22例(91.7%)和2例(8.3%)。见表2。表2中同时列出了相关文献检测到的同一地区健康人群中EBV 1/2分型和F/f分型的结果[16,18]。白血病、MDS以及健康人群中1/2分型的分布差异无显著性(P>0.05),F/f分型的分布差异亦无显著性(χ2=3.030,P>0.05)。
2.3 EBERs基因的序列变异
本文40例白血病和20例MDS标本EBERs基因相应片段扩增成功,其变异情况见图1。根据相关文献分类方法[18],检测到两种EBERs基因型:EB-6m和B95-8。11例标本为B95-8型序列,包括6例(15.0%)白血病标本和5例(25.0%)MDS标本,B95-8型序列与EBV标准株序列相同或仅出现散在无规律突变,见图1。49例标本为EB-6m型,包括34例(85.0%)白血病和15例(75.0%)MDS标本,见表2。与B95-8比较,EB-6m具有6处共有突变,除1处(7123nt A→G)位于EBER2基因,其余5处均位于EBER1和EBER2基因之间161 bp(nt6796~6955)间隔序列中,分别为6808nt T→A、6884nt G→A、6886nt T→G、6911nt A→G和6944nt G→A。其中1例MDS标本中EB-6m亚型只有5个共有突变序列(6808 nt、6884 nt、6911 nt、6944 nt和7123 nt)。除共有突变外,部分样本还出现其他位点的散在突变(图1)。EBERs变异类型在白血病和MDS中分布差异无显著性(P>0.05),但白血病、MDS與以往同一地区健康人群之间的EBERs基因变异类型的分布不同,差异有统计学意义(χ2=5.455、8.706,P<0.01)。 圖中第2行数字对应于核苷酸位点,B95-8核苷酸序列在第2行下面。第1列为标本类型,第2列为不同的基因亚型;图中第3列把相同序列的标本合并,以1例标本为其代表株,标本编号后括号内数字为该相同序列的标本数。图中大写字母表示核苷酸。
3 讨 论
EBV可能在白血病和MDS发病中发挥着重要的作用[21-23]。本文的研究结果显示,186例白血病样本中有69例(37.1%)、77例MDS样本中有28例(36.4%)为EBV阳性标本。白血病和MDS病人中EBV检出率高于健康对照组(5.4%,2/37)[8]。
本研究结果显示,白血病和MDS中1型EBV检出率分别为77.3%(51/66)和89.3%(25/28),F型EBV在白血病和MDS中的检出率分别为98.6%(68/69)和91.7%(22/24),提示山东地区白血病和MDS均以1型和F型EBV为主,这与以往该地区鼻咽癌、胃癌、淋巴瘤和健康人群的研究结果一致,也与世界其他多数地域的研究结果一致[17-18]。以往研究显示,1型EBV是大多数地区的优势毒株,而2型EBV仅在中非和新几内亚占主导地位[24-25];F型EBV是除我国南方地区鼻咽癌高发区以外地域的主要基因型,f型EBV仅在我国南方鼻咽癌高发区较为多见,而且其在高发区鼻咽癌中的检出率高于非鼻咽癌人群,被认为与高发区鼻咽癌相关[18]。
以往研究发现了4种EBERs基因型:EB-6m、EB-8m、EB-10m和B95-8,山东地区EBV相关胃癌、鼻咽癌、淋巴瘤和健康人群以及广东地区健康人群均以EB-6m变异型为主,而EB-8m变异型仅为中国南方鼻咽癌的主要变异型,且其检出率高于健康人群,可能与鼻咽癌尤其是高发区鼻咽癌发生相关[16-18]。已有研究显示,美国的16例霍奇金病有13例(81.3%)EBERs基因型为EB-6m[26],3例EBERs基因型为B95-8;24例移植后淋巴增殖性疾病有19例(79.2%)EBERs基因型为EB-6m[27],5例EBERs基因型为B95-8;德国的41例良性和恶性疾病中有31例(75.6%)EBERs基因型为EB-6m,10例EBERs基因型为B95-8[28];意大利的15例人类免疫缺陷病毒感染的病人有14例(93.3%)EBERs基因型为EB-6m,1例EBERs基因型为B95-8,11例健康人群有7例(63.6%)EBERs基因型为EB-6m,4例EBERs基因型为B95-8[29];南非21例鼻咽癌有18例(85.7%)EBERs基因型为EB-6m,3例EBERs基因型为B95-8[30]。由上述数据可见,EBERs变异除与地理分布有关外,可能还具有疾病关联性。
本研究结果显示,EBERs基因EB-6m亚型是白血病和MDS中最常见的类型,而未发现EB-8m和EB-10 m亚型;白血病和MDS中EBERs基因型EB-6m检出率分别为85.0%(34/40)和75.0%(15/20),低于健康人群(96.7%,89/92),本文结果与以往其他人群中的研究结果相一致[16-17]。因此,EBERs变异在白血病和MDS发病中的作用可能与其他EBV相关肿瘤相同,EB-6m型EBERs可能与白血病及MDS的发生相关,但这需要来自更多地域人群的相关研究加以证实。EBERs是非编码RNA,EBERs可通过促进细胞生长和调节免疫功能而参与鼻咽癌和Burkitt淋巴瘤等肿瘤的发生发展[31]。同时,EBV编码的小RNA(EBERs)可以诱导自分泌生长因子的表达,维持Burkitt淋巴瘤细胞的恶性表型,表明EBERs在肿瘤发生中有潜在作用[32]。近年来,EBV阳性淋巴瘤、胃癌及鼻咽癌细胞系中EBERs在细胞转化中的作用已经被证实[31]。白血病和MDS的发生可能与EBERs在参与免疫调节中某些特异蛋白的表达有关。因此,有必要对EB-6m变异型EBERs及含EB-6m变异型EBERs的EBV毒株进行功能学研究,探讨该变异对致癌潜能以及免疫调节的影响,这对最终阐明EBERs基因变异在白血病和MDS发生中的作用具有重要意义。
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