成人EB病毒相关淋巴细胞增殖病克隆性转化的研究

Lymphocytic Clonal Expansion in Adult Patients with Epstein-Barr Virus-Associated Lymphoproliferative Disease

目的:探讨成人EB病毒相关淋巴细胞克隆性转化的实验方法,为EB病毒相关淋巴细胞增殖病的早期诊断、分类、预测转归提供客观的判断指标。方法:采集5例成人EB病毒相关的淋巴组织增殖性疾病(EBV+LPD)患者的外周血标本,另采集4例成人传染性单核细胞增多症(IM)外周血标本作为阴性对照,3例急性NK细胞白血病(ANKL)的外周血标本作为阳性对照。应用流式细胞术(FCM)检测淋巴细胞免疫表型、RT-PCR检测T细胞受体(TCR)基因重排,Southern blot方法鉴定EBV末端重复序列(TR)多态性,对患者外周淋巴细胞进行了克隆性分析。结果:在5例EBV-LPD患者中,FCM仅检测出1例TCRVβ表型呈克隆性;RT-PCR检测TCRVβ时在IM患者均检测出TCR受体基因重排阳性,在5例EBV-LPD患者中检测出4例阳性;在5例EBV+LPD患者中1例检测有单克隆条带,2例检测有寡克隆条带。结论:流式细胞术表型分析的敏感性较差;TCRVβ重排不能区分反应性克隆和转化型克隆;而EB病毒末端重复序列克隆性鉴定的方法,能够更客观特异地反应EB病毒相关淋巴细胞增殖的克隆性转化,有助于改善疾病的早期诊断、分类及预测临床转归。

Objective： To explore the lymphocytic clonal expansion in adult patients with Epstein-Barr virus-associated lymphoproliferative diseases（ EBV ＋ LPD）,and to investigate the experimental methods for EBV＋LPD cells so as to provide a more objective measure for the diagnosis,classification and prognosis in the early stage of this disease. Methods：Peripheral blood samples from 5 patients with EBV ＋ LPD,4 patients with adult infectious mononucleosis（ IM） as negative control and 3 patients with acute NK-cell leukemia（ ANKL） as positive control were collected. Prior to immunochemotherapy,viral loads and clonality were analysed by flow cytometry（ FCM）, T cell receptor gene rearrangement（ TCR） was detected by real-time polymerase chain reaction（ RT-PCR）,and diversity of EB virus terminal repeat（ EBV-TR） was detected by Southern blot. Results： FCM showed only 1 case with clonal TCRVβ in 5 patients with EBV ＋ LPD,TCR clonal expansion could be detected both in patients with IM（ 4 of 4） and 4 patients with EBV ＋ LPD（ 4 of5）,Out of patients with EBV ＋ LPD,1 patient displayed a monoclonal band and 2 patients showed oligoclonal bands when detecting EBV-TR by southen blot. Conclusion： Detecting the diversity of EBV-TR by Southern blot may be the most objective way to reflex clonal transformation of EBV ＋ LPD,which is of great benefit to the diagnosis,classification and prognosis in the early stage of this disease.