Borna病病毒与慢性粒细胞白血病:病例对照  

Borna disease virus and chronic myelocytic leukemia:A case- control study

作　　者：赵立波[1,2] 谢鹏[1,2] 牟君[1,2] 杨泽松[3] 李亚军[1,2] 邹德智[1,2] 刘庆军[1,2] 

机构地区：[1]重庆医科大学附属第一医院神经内科 [2]重庆市神经病学重点实验室,重庆市400016 [3]重庆医科大学附属第一医院血液科

出　　处：《中国临床康复》2006年第21期15-18,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金(30470605)~~

摘　　要：目的:检测人类慢性粒细胞白血病中Borna病病毒的感染率情况,分析Borna病病毒与慢性粒细胞白血病是否存在相关性。方法:选取2004-01/2005-06重庆医科大学附属第一医院血液科收治的符合细胞形态学、免疫学及遗传学分型诊断标准的30例慢性粒细胞白血病确诊患者,另以30例健康自愿献血者作为正常对照。慢性粒细胞白血病患者和正常对照人群均取10mL外周枸橼酸二钠抗凝血,采用Ficoll-conray液分离出外周血单个核细胞,提取RNA,随后采用紫外分光光度计测A260,A280值,计算RNA浓度。采用琼脂糖凝胶电泳进行RNA完整性检测,建立Borna病病毒P24阳性定量标准曲线,随机抽取一浓度质粒聚合酶链反应扩增产物进行克隆测序。采用荧光定量套式反转录聚合酶链反应检测RNA中Borna病病毒P24基因片段。结果:①5个梯度阳性模板定量扩增后均呈典型的S形,起始模板浓度与循环阈值相关系数达0.99969,具有良好的线性关系。质粒PCR产物克隆测序结果与美国国立卫生研究院基因列数据库中调出Borna病病毒的第二开放阅读框(ORFⅡ)区P24序列一致,确定扩增成功。②4份样本电泳后可见28s,18s两条清晰的条带和稍欠清晰的5s条带。A260/A280值分别为1.825,1.946,1.833,1.895,均在1.8以上。RNA浓度分别为0.584,0.872,0.416,0.524mg/L。RNA的提取质量达到聚合酶链反应要求。③电泳条带清晰,反转录及第1轮聚合酶链反应扩增成功。④第2轮聚合酶链反应阳性模板扩增荧光呈典型的S形,说明定量聚合酶链反应扩增成功,起始模板浓度与循环阈值相关系数达0.99以上,具有良好的线性关系。慢性粒细胞白血病患者Borna病病毒P24基因片段与正常对照者的阳性率均为0%,无差异。结论:本实验不支持Borna病病毒与慢性粒细胞白血病的发生具有相关性。AIM： To detect the infection of Borua disease virus （BDV） in the human chronic myelocytic leukemia （CML） and analyze whether BDV is linked to CML METHODS： Totally 30 patients who were diagnosed as CML, met the diagnostic standard of cell morphology, immunology and genetics and received treatment in the Department of Hematology, First Hospital Affiliated to Chongqing Medical University from January 2004 to June 2005 were recruited, and another 30 healthy volunteers who donated their blood as normal control. According to the specific sequence of BDV p24 gene, the primers and the fluorescene probe were synthesized and designed, All things involved in the reverse transcriptase polymerase chain reaction were treated with ribonuclease inhibitor. A 10 mL EDTA-blood was collected from peripheral vein of each patient and healthy donor. Peripheral blood mononuclear cells was isolated through Ficoll-conray, then RNA was extracted from the peripheral blood mononuclear cells. Then the concentration of the RNA were checked with ultraviolet spectrophotometer through measuring their value of A260 and A280 and their integrality were analyzed by electrophoresis in an agarose gel. The quantitative standard curve of BDV P24 was successfully established. The product of one plasmid taken out from the five plasmids at randomly was cloned and its sequence was analyzed. The P24 fragment of BDV in RNA was detected by fluorescence quantitative nested reverse transcriptase polymerase chain reaction （FQ-nRT-PCR）. RESULTS： ①Five gradient positive templets presented typical S shape after quantitatively enlarged. The relative coefficient of initial templet concentration and circulated threshold reached 0.99969 with good linear relationship. Plasmid PCR product clone sequencing results is consistent with P24 sequencing of the second open reading frame （ORF Ⅱ ）district of Borna disease virus in gene sequencing database of National Institutes of Health, which determining the amplification is successful. ② Electropho

关 键 词：白血病 髓样 慢性 Boma病病毒/遗传学 逆转录聚合酶链反应 

分 类 号：R733.7[医药卫生—肿瘤]