【作者】 顾斌；

【导师】 吴德沛； 陈广华；

【作者基本信息】 苏州大学 ， 血液病学， 2013， 硕士

【摘要】 第一部分：EBV特异性细胞毒性T淋巴细胞体外诱导培养及杀伤效果鉴定目的研究EB病毒特异性细胞毒性T淋巴细胞(EBV-CTL)体外诱导和扩增培养的方法，并检测其杀伤的效果。方法采集EBV血清抗体阳性的6例正常供体的外周血单个核细胞（PBMNC），用EBV转化的B淋巴细胞系（BLCL）作为抗原递呈细胞及抗原刺激剂,经辐照灭活后不断刺激培养自体PBMNC，诱导产生EBV-CTL，并将其扩增培养；采用流式细胞仪鉴定其免疫表型；然后检测不同效靶细胞比例条件下EBV-CTL对自体BLCL、自体植物血凝素培养的B淋巴母细胞（PHA-Blast）、HLA不合供体的BLCL（alloBLCL）、K562细胞株的杀伤效果。结果6例EBV血清抗体阳性正常供体来源的PBMNC在体外成功筛选并扩增培养了EBV-CTL，扩增效率为PBMNC数的18.6~55.0倍。刺激10次培养后的EBV-CTL对自体BLCL在20:1，10:1，5:1三种效靶细胞比例条件下特异性杀伤效率的平均值分别为59.4%、43.2%、29.0%；对PHA-Blast、alloBLCL、K562的非特异性杀伤率在上述三种效靶细胞比例下平均值依次为7.1%、9.4%、10.3%（p﹤0.05），6.6%、8.3%、8.1%（p﹤0.05），5.4%、7.3%、6.3%（p﹤0.05）。结论EBV-CTL能有效在体外筛选培养并扩增，并能有效杀伤HLA相合的BLCL；可望成为EBV相关性移植后淋巴细胞增殖性疾病的过继免疫治疗的有效方法。第二部分：异基因造血干细胞移植后淋巴细胞增殖性疾病的临床分析目的提高对异基因造血干细胞移植后淋巴细胞增殖性疾病（PTLD）的认识，探讨其有效的治疗策略。方法回顾性分析2006年1月至2011年12月我院行异基因造血干细胞移植的524例病例，其中同胞全相合移植299例，无关供体相合移植149例，亲缘半相合移植51例，脐血移植25例。结果根据临床表现及病理结果共确诊7例PTLD；在上述四种移植类型中PTLD发病率依次为0.3%（1/299），2.7%（4/149），2.0%（1/51），4.0%（1/25）；后三者发病率均高于同胞全相合移植组（P=.044）；在预处理应用抗胸腺细胞球蛋白（ATG）的患者中发病率为2.7%（6/225），明显高于无ATG者（1/299, P=.046）。7例PTLD临床表现特征主要分为两类：6例发生在移植后2至8个月，播散性起病，正规经验性抗感染治疗无效的发热，淋巴结进行性肿大，血象三系进行性下降，EB病毒血症，病情迅速进展，短期内出现多脏器功能不全；1例发生在移植后18月，以颌下淋巴结进行性肿大局限性起病，EBV-DNA检测阴性，病程相对缓和。病理类型6例为弥漫大B细胞淋巴瘤，1例为小淋巴细胞性淋巴瘤。7例中4例给予含利妥昔单抗方案治疗，2例恢复，2例死于PTLD进展；1例给予供者淋巴细胞输注后恢复；其余2例对抗病毒对症支持治疗无效死亡。结论PTLD是一组异质性淋巴细胞增殖性疾病，尽早诊断，及时采用有效措施治疗有望改善其预后。更多还原

【Abstract】 Part1:Ex vivo cultrued Epstein-Barr virus specific cytotoxic T lymphocytes andevaluation of the killing effectObjective To explore the method for induction and expandsion of EB virus specificcytotoxic T lymphocytes (EBV-CTL) in vitro, and detect the killing effect. MethodsPeripheral blood mononuclear cells (PBMNC) were collected from six EBV seropositivehealthy donors, and EBV-transformed B lymphoblastoid cells (BLCL) were used as theantigen-presenting cells and antigen stimuli which was irradiated by40Gy60Co. Theautologous PBMNCs and irradiated BLCLs were cultured to induce and expand theEBV-CTLs, and the immunophenotype was identified by the flow cytometry. The killingeffect of the EBV-CTLs against the autologous BLCLs (autoBLCL), the autologous PHAcultured B lymphoblastoid cells (PHA-BLCL), the allogeneic BLCL (alloBLCL) and theK562cell strain were measured with LDH release assay under different effector-to-targetratio. Results Six cell lines of EBV-CTLs were induced and expanded from the EBVseropositive healthy donors, the overall increase in cell numbers varied from18.6to55.0times. After10times stimulations, the specific killing efficiency of the EBV-CTLs for theautoBLCL were59.4%,43.2%and29.0%under the effector-to-target ratio of20:1,10:1and5:1. The nonspecific killing efficiency for the PHA-Blast, alloBLCL and K562cellswere7.1%,9.4%and10.3%(p﹤0.05) under the20:1ratio;6.6%,8.3%and8.1%(p﹤0.05) under10:1;5.4%,7.3%and6.3%(p﹤0.05) under5:1, respectively. ConclusionEBV-CTL can be successfully induced and expanded ex vivo for specific killing of HLAmatched BLCLs, as could be the potential treatment for EBV related post-transplant lymphoproliferative disorders.Part2:Clinical analysis of lymphoproliferative disorders after allogeneichematopoirtic stem cell transplantationObjective To study the early diagnosis of post-transplantation lymphoproliferativedisorders (PTLD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) andinvestigate the effective therapy strategy. Methods From January2006to December2011,524cases of hematologic disease treated with allo-HSCT were analysedretrospectively, including299related matched donors,149unrelated volunteer donors,51related haploidentical donors,25unrelated cord blood at our hospital. Results Sevencases were diagnosed PTLD by the clinical manifestations and pathology. PTLD incidencerates of these four types of transplants were0.3%(1/299),2.7%(4/149),2.0%(1/51),4.0%(1/25), respectively; the incidence rates of the last three were significantly higher than thefirst one (P=.044); the incidence rate in patients with conditioning regimen includingantithymocyte globulin (ATG) was significantly higher than that without ATG(2.7%vs0.5%, P=.046). The clinical features of the seven cases could be divided into twocategories: six cases occurred during2-8months after transplantation with disseminatedonset, recurrent high fever, lymph node swelling, decline in the number of three lineages ofperipheral blood, EB viremia and failure response to formal empirical anti-infectivetherapy, then the disease rapidly deteriorated in the short term. One case manifested ineighteen months after transplantation with located submandibular lymph node enlargement,negative EB viremia, and the course was relatively mild. Pathological types of six caseswere diffuse large B-cell lymphomas, and one small lymphocytic lymphoma. Four ofseven cases treated with rituximab, two survived and two died of PTLD progression; onewas treated with donor lymphocyte infusion and got recovery; the remaining two caseswith anti-viral and supportive therapy died soon. Conclusions PTLD is a heterogeneousgroup of lymphoproliferative disorders, early diagnosis and timely treatment with theeffective measures are expected to improve the prognosis.更多还原