抑制性杀伤细胞受体在NK细胞和T细胞上的表达及其与急性移植物抗宿主病的关系

 目的 研究异基因造血干细胞移植（allo-HSCT）患者移植前后外周血NK及T细胞上4种抑制性杀伤细胞受体（CD158a、CD158b、NKB1和CD94／NKG2A）的表达及其与急性移植物抗宿主病（aGVHD）的关系。方法 采用流式细胞术检测NK及T细胞上抑制性杀伤细胞受体的表达。结果 NK细胞上CD158a和CD158b的表达于移植后3～4个月、NKB1和CD94／NKG2A的表达于移植后2个月恢复移植前水平。移植前后CD158a和NKB1在CD+3 T细胞上持续低水平表达;移植前CD158b和CD94／NKG2A在CD+3 T细胞上的表达水平较高,且主要表达于CD+8 T细胞上,移植后其在CD+8 T细胞上的表达增加。CD+8 T细胞上CD158b的表达在发生Ⅰ度aGVHD时显著增高,与无aGVHD组及Ⅱ~Ⅳ度aGVHD组相比,差异均有统计学意义（P均＜0.05）;在发生Ⅱ~Ⅳ度aGVHD时增高不明显,与无aGVHD组相比,差异无统计学意义（P＞0.05）。结论 CD158b在CD+8 T细胞上高表达可能有助于降低T细胞同种反应性,减轻aGVHD的程度。     

异基因造血干细胞移植后免疫重建的临床研究及急性移植物抗宿主病的危险因素分析

目的 探讨异基因造血干细胞移植后患者外周血淋巴细胞亚群表达水平与急性移植物抗宿主病(aGVHD)的相关性,探究移植后免疫重建的一般规律及引起aGVHD的危险因素.方法 回顾性分析2012年1月到2013年11月行异基因造血干细胞移植的65例患者临床资料,应用流式细胞术检测患者移植后1、3、6、12个月外周血淋巴细胞亚群表达水平,研究免疫重建规律,分析淋巴细胞亚群与aGVHD的相关性;运用Logistic回归分析aGVHD的危险因素.结果 移植后1个月CD3+比例下降,移植后3个月回升至正常,平均为(63.65±6.64)％;其中,CD3+ CD4+比例在移植后降低,移植后12个月恢复至正常,达(23.94±3.15)％;而CD3+ CD8+比例恢复较快,移植后1个月即接近正常,达(31.06±8.40)％;CD3+ CD4+/CD3+ CD8+比值在移植后处于低水平,后缓慢回升,但移植后12个月时仍处于倒置,为0.54+0.11.CD3+ CD25+比例在移植后1个月低于正常水平,为(2.16±0.93)％,3个月恢复正常;CD3-CD15+ CD56+比例在移植后1个月明显升高,达(42.31±8.47)％,移植后6个月逐渐回落至正常.移植后的前3个月,Ⅲ～Ⅳ度aGVHD组的CD3+比例高于无aGVHD组(1个月时,P=0.037;3个月时,P=0.045),CD3+ CD25+比例高于无aGVHD组(1个月时,P=0.025;3个月时,P=0.041),CD3+ CD4+/CD3+ CD8+比值低于无aGVHD组(1个月时,P=0.040;3个月时,P=0.043).移植后1个月无aGVHD组的CD3+ CD4+比例高于Ⅲ～Ⅳ度aGVHD组(P=0.032),CD3-CD15+ CD56+比例高于Ⅲ～Ⅳ度aGVHD组(P=0.046),CD3+ CD8+比例低于Ⅲ～Ⅳ度aGVHD组(P=0.029).供受体HLA配型不全合(OR =2.511,P=0.024)、无关供体(OR=2.964,P=0.018)、不使用抗胸腺细胞球蛋白(ATG)预防GVHD(OR=2.792,P=0.033)是发生aGVHD的危险因素.结论 移植后早期的T细胞免疫重建主要以非胸腺依赖性的CD8+细胞为主,而胸腺功能的损伤可引起依赖胸腺途径再生的T细胞(如CD4+细胞)重建迟缓.CD25+T细胞与aGVHD有密切关联,其表达水平与aGVHD的严重程度呈正相关.供受体HLA配型不全合、无关供体、不使用ATG预防是发生aGVHD的危险因素.     

苦参碱诱导自然杀伤细胞对白血病K562细胞杀伤的实验研究

目的 探讨中药苦参碱对人自然杀伤（NK）细胞体外杀伤白血病细胞的作用及可能的分子机制.方法 以人慢性粒细胞白血病K562细胞为靶细胞,采用CFSE/PI双染色法流式细胞术检测不同质量浓度（02、0.5、0.8 mg/ml）苦参碱处理后,人NK细胞在不同效靶比下对K562细胞的体外杀伤活性.流式细胞术分析不同浓度苦参碱处理24 h对NK细胞主要活化性受体NKG2D和抑制性受体CD158a、CD158b表达的影响及K562细胞膜上NKG2D配体MICA/B、ULBP1、ULBP 2、ULBP 3表达的改变.结果 效靶比为5∶1时,NK细胞对0.2、0.5和0.8 mg/ml苦参碱处理后的K562细胞杀伤率分别为32.8％、38.1％和40.5％,较处理前均有不同程度增高（29.2％）;但进一步增加效靶比（10：1）后,NK细胞杀伤活性改变差异无统计学意义（P＞0.05）.苦参碱处理24 h,NK细胞抑制性受体CD158a、CD158b的表达均较处理前降低,而活化受体NKG2D的表达则增高.K562细胞表面NKG2D配体ULBP1和ULBP2分子的表达也较处理前增高（平均荧光强度分别为174.33±39.93比275.67±32.88,517.6±47.97比1368.6±49.43,P＜0.05）.结论 苦参碱可增强NK细胞对白血病K562细胞的体外杀伤活性,其机制可能与NK细胞受体及配体表达调节作用有关.     

乳腺癌患者可溶性MICA对自然杀伤细胞受体的影响

目的观察乳腺癌患者外周血自然杀伤（NK）细胞杀伤活性及受体的变化,探讨可溶性MICA（sMICA）对NK细胞受体及杀伤活性的影响。方法ELISA法检测外周血血清sMICA的含量。流式细胞术（FCM）检测NK细胞百分比、NK细胞活化性受体NKG2D、抑制性受体KIR（CD158b）表达。MTT法检测NK细胞对乳腺癌细胞株MCF-7的杀伤活性。结果与健康人比较,乳腺癌患者中81．6％表达sMICA,含量为（205．36±71．27）ng／L,且sMICA含量与TNM分期呈正相关。乳腺癌患者外周血NK细胞所占百分比无明显差异,但血清sMICA阳性的乳腺癌患者中NK细胞杀伤活性明显降低,NKG2D表达下降,CD158b表达增高。当NK细胞培养体系中加入sMICA阳性的乳腺癌血清时,其杀瘤活性明显降低【（76．2±6．7）％与（48．4±4．1）％】,NKG2D的表达明显下调[（92．5±7．1）％与（62．5±6．4）％],而CD158b的表达明显上升【（10．6±3．2）％与（43．6±3．4）％】。sMICA阳性的乳腺癌患者NK细胞与细胞因子IL-15共培养,NK细胞的杀瘤活性、NKG2D的表达明显升高,KIR（CD158b）的表达明显下降。结论乳腺癌外周血血清中sMICA可通过下调NK细胞NKG2D表达以及上调KIR表达,降低NK细胞杀瘤活性。IL-15可逆转sMICA对NK细胞的免疫下调作用。     

CD4^＋CD25^High调节性T细胞及负调控因子白细胞介素-10与异基因造血干细胞移植后移植物抗宿主病的关系研究

目的 研究异基因造血干细胞移植（allo-HSCT）后患者外周血CD4^＋CD25^High调节性T细胞（Treg细胞）及细胞因子白细胞介素-10（IL-10）与急性移植物抗宿主病（aGVHD）的关系.方法 采用流式细胞术检测13例allo-HSCT后患者外周血CD4^＋CD25^HighFoxp3^HghTreg细胞、CD4^＋CD25^HighCD^127LowTreg细胞占CD^4＋T细胞的百分含量,同时用酶联免疫吸附法（ELISA）检测对应时期血清中IL-10的质量浓度.结果 13例患者均获得造血功能重建;aGVHD组CD4^＋CD25^HighCD127^Low/CD4^＋与CD4^＋CD25^HighFoxp3^High/CD4^＋比例均明显低于无GVHD组,差异有统计学意义（P＜0.01）;Ⅲ～Ⅳ度aGVHD亚组CD4^＋CD25^HighCD^127Low/CD4^＋与CD4^＋CD25^HighFoxp^3High/CD4^＋比例低于Ⅰ～Ⅱ度aGVHD亚组,但差异无统计学意义（P＞0.05）;相同组别间CD4^＋CD25^HighCD^127Low/CD4^＋与CD4^＋CD25^HighFoxp^3High/CD4^＋差异无统计学意义.aGVHD组的IL-10质量浓度明显低于无GVHD组,差异有统计学意义（P＜0.01）.Treg细胞与IL-10变化呈相关性（r=0.925,P＜0.05）.结论 Treg细胞的水平与allo-HSCT后aGVHD的发生有密切关系;通过监测Treg细胞水平对临床早期诊断aGVHD及判断aGVHD预后、指导免疫调节剂的应用具有重要意义;CD127^Low可以作为Treg细胞表面的特异标志,推进对Treg细胞的检测及分离纯化;IL-10是一种重要的负调控因子;Treg细胞与IL-10的表达在aGVHD患者存在相关性,可能为Treg细胞的免疫抑制机制提供一定基础.     

单倍体造血干细胞联合脐血输注治疗白血病的临床研究

目的 分析单倍体造血干细胞联合第三方脐血输注治疗白血病的疗效及安全性.方法 对苏州大学附属第一医院2011年1月至2012年5月44例采用单倍体造血干细胞联合第三方脐血输注治疗的白血病患者进行回顾性分析,其中急性淋巴细胞白血病（ALL） 16例、急性髓系白血病（AML）24例、慢性粒细胞白血病（CML）3例、T淋巴母细胞淋巴瘤1例.结果 44例白血病患者回输的单倍体供体移植物中CD34＋细胞中位数为3.14×106/kg（1.68×106/kg ～ 8.32× 106/kg）,回输的第三方脐血中CD34＋细胞中位数为1.03×105/kg（0.31×105/kg～5.32×105/kg）.42例（95.45％）患者造血重建,中性粒细胞造血重建时间为14d（10～29 d）,血小板造血重建时间为23d（11～89d）.对42例植入成功的患者长期监测供受体嵌合率（STR）,除5例（11.90％）患者在移植后3～13个月复发,其余37例（88.10％）患者STR均≥95％.17例（40.47％）患者发生不同程度的急性移植物抗宿主病（aGVHD）,发生Ⅱ～Ⅳ度aGVHD的患者共8例（19.05％）,发生Ⅲ～Ⅳ度aGVHD的患者为4例（9.52％）.23例（54.76％）发生慢性移植物抗宿主病（cGVHD）,其中局限型17例（40.47％）,广泛型6例（14.29％）.1年总生存（OS）率为70.84％,1年无事件生存（EFS）率为63.34％.结论 初步研究证实单倍体造血于细胞联合第三方脐血输注治疗白血病安全有效,从而为异基因造血干细胞的移植提供了新的方法.     

阻断诱导型共刺激分子信号通路对小鼠急性移植物抗宿主病的影响

目的:观察以诱导型共刺激分子(inducible costimulator,ICOS)融合蛋白阻断ICOS-B7H途径对小鼠急性移植物抗宿主病(acute graft-versus-host disease,aGVHD)的作用.方法:建立以C57 BL/6鼠为供体、致死剂量照射的BALB/c鼠为受体的aGVHD模型,分别在移植0、2、4、8d后腹腔注射100 μg的ICOS-Ig,以注射同剂量的人Ig(h-Ig)作为对照.观察小鼠体重、生存率及临床GVHD症状的改变;移植4d后取小鼠脾脏单个核细胞,流式细胞术检测H-2Kd - CD4+及H-2Kd - CD8+细胞的CFSE强度,确定供体T淋巴细胞的增殖率;移植10 d后采用Annexin-V及PI试剂盒检测供体H-2Kd- CD4+及H-2Kd -CD8+细胞的凋亡率.结果:ICOS-Ig干预可有效减轻小鼠aGVHD的严重程度,ICOS-Ig组小鼠的生存时间比h-Ig组显著延长[(20.0±3.1) vs (10.0±2.1)d,P=0.0217,n=14],其肠道黏膜病变及肝脏汇管区淋巴细胞浸润明显减少.移植3d后,ICOS-Ig组和h-Ig组小鼠的CD4+ CFSE -和CD8+ CFSE -细胞群比例无明显差异[ (98.88±0.76)％ vs (99.71 ±0.27)％,(98.62±0.63)％vs(99.53±0.51)％,P＞0.05];ICOS-Ig增加了体内异基因CD8+T淋巴细胞的凋亡率[(15.7±9.59)％vs(25.92±5.66)％,P=0.032],不影响异基因CD4+T细胞的凋亡率[(15.72±7.34)％ vs (22.78±6.94)％,P=0.078].结论:阻断ICOS-B7H通路可促进活化的T淋巴细胞凋亡,减轻造血干细胞移植治疗血液肿瘤中发生的aGVHD的严重程度.     

肺癌患者调节性T细胞与NK细胞活化受体NKG2D的相关性及其临床意义

目的 分析肺癌患者外周血中CD+4 CDHi25 CDLo127调节性T细胞及自然杀伤细胞(NK细胞)活化受体NKG2D的表达水平之间的关系,探讨其在肿瘤免疫逃逸机制中的作用及临床意义.方法 选择70例肺癌患者,均经病理确诊.采用流式细胞术(FCM)检测患者外周血中CD+4 CDHi25 CDLo127调节性T细胞、NK细胞及NKG2D表达水平,并以50名健康人为对照.结果 肺癌患者外周血中CD+4 CDHi25 CDLo127调节性T细胞比例较健康对照组明显升高[(8.4±4.1)％与(6.7±1.7)％],差异有统计学意义(t=3.09,P＜0.05);肺癌组NK细胞比例与健康对照组比较[(15.6±8.3)％与(17.2±4.2)％],差异无统计学意义(t=-1.33,P＞0.05);肺癌组NKG2D较健康对照组明显降低[(83.3±4.9)％与(87.4±2.9)％],差异有统计学意义(t=3.16,P＜ 0.05).CD+4 CDHi25 CDLo127调节性T细胞与NKG2D呈负相关性(r=-0.302,P＜0.05).结论 在肺癌患者外周血中调节性T细胞可能通过下调NKG2D,参与肿瘤免疫逃逸机制,二者可作为评估肺癌患者免疫功能状态及预后的参考指标.     

供者细胞回输数量对单倍型造血干细胞移植后急性移植物抗宿主病的影响

目的 评价单倍型造血干细胞移植(Haplo-HSCT)后重度急性移植物抗宿主病(aGVHD)发生率与回输细胞数量的关系.方法 回顾性分析2009年1月至2013年12月进行Haplo-HSCT的68例患者临床资料,分析移植前后各因素与移植后Ⅲ~Ⅳ度aGVHD发生率的关系.结果 68例患者中,男性42例,女性26例,HLA配型为5/10至9/10相合,其中父亲供者19例,母亲供者24例,兄弟姐妹供者16例,子女供者9例.未出现Ⅲ~Ⅳ度aGVHD患者51例,其中男性32例,女性19例,中位年龄20岁(5~55岁);出现Ⅲ~Ⅳ度aGVHD患者17例,其中男性10例,女性7例,中位年龄23岁(5~54岁).两组回输的单个核细胞(MNC)计数、CD34+细胞数以及移植后白细胞计数和血小板计数恢复快慢程度差异均无统计学意义(均P>0.05),以上4个因素中MNC与CD34+细胞数量、白细胞植活时间存在相关性(均P<0.05),而CD34+细胞数量及白细胞植活时间均与血小板植活时间有相关性(均P<0.05).结论 移植后发生Ⅲ~Ⅳ度aGVHD与回输的MNC及CD34+细胞数量无关.     

脐血免疫细胞特性的研究

目的：研究单份脐血免疫细胞的表型特征。方法：收集20份脐血，采用6％的羟乙基淀粉沉淀去除红细胞，用流式细胞仪检测脐血免疫细胞的表型特征。结果：CD4+T细胞表达CD(45RA)为(89.95±7.86)％，CD8+T细胞表达CD(45RA)为(99.58±3.46)％，均显著高于成人外周血T淋巴细胞(P＜0.01)。结论：脐血中免疫细胞发育不成熟，免疫原性弱，用于移植时移植物抗宿主病(GVHD)的发生率较骨髓移植(BMT)和自体成人外周血造血干细胞移植(PBSCT)明显降低。     

Nonmyeloablative hematopoietic cell transplantation

The myeloablative doses of chemotherapy and radiation used with conventional allogeneic hematopoietic cell transplantation produce considerable morbidity and mortality that generally limit this treatment to patients younger than 55 years of age and in good general medical condition. It has become clear that T-cell-mediated graft-versus-tumor effects play an important role in the elimination of malignant disease after allotransplants. Several investigators have sought to reduce regimen-related toxicities while optimizing graft-versus-tumor effects. Strategies can be broadly categorized as reduced-intensity regimens that retain some toxicities and require hospitalization, and minimally myelosuppressive regimens that rely on immunosuppression for allogeneic engraftment and resultant graft-versus-tumor effects. The latter approach can be performed in the ambulatory care setting. Preliminary results are encouraging. If long-term efficacy is demonstrated, such strategies would expand treatment options for patients who would otherwise be excluded from receiving conventional allografts.     

Usefulness of hematopoietic cell transplantation-specific comorbidity index after allogeneic hematopoietic stem cell transplantation

We retrospectively investigated the hematopoietic cell transplantation-specific comorbidity index(HCT-CI)to predict non relapse mortality. Of 127 patients who underwent transplantation between January 2000 and December 2003 with conditioning consisting of total body irradiation, cyclophosphamide and thiotepa, HCT-CI scores were obtained for 83 patients. Median age was 42 years. The sources of stem cells included HLA-identical bone marrow or peripheral blood from sibling(30), HLA-matched bone marrow from unrelated donors(45), and HLA-mismatched bone marrow or peripheral blood from family donors(8). Hematological disease was divided into two groups, standard risk(47)and high risk(36). Standard risk indicates acute leukemia in first or second remission and chronic myelocytic leukemia in first chronic phase, while high risk indicates all other diagnoses. There were 45 patients with moderate or severe pulmonary comorbidities. 55 patients with HCT-CI scores of 2 or less had higher 2-year overall survival than 28 patients with HCT-CI scores of 3 or more(65% vs. 36%, p=0.0009). Although the non relapse mortality rate was not different, HCT-CI scores were a more useful indicator to predict survival in high risk patients than in standard risk patients. Prospective evaluation is warranted to clarify the usefulness of HCT-CI.     

Myelodysplastic syndrome hematopoietic stem cell

Myelodysplastic syndromes (MDSs) are clonal hematopoietic stem cell (HSC) malignancies that are characterized by ineffective hematopoiesis and frequent progression to acute myeloid leukemia (AML). Thus far, few treatments can actually alter the natural history of this disease. Allogeneic stem‐cell transplantation for high‐risk MDS is becoming the only curative therapy probably because of the improvement of bone marrow transplant procedures. The lack of other options underscores the urgent need to develop new therapy. The prevailing model suggests that genetic and/or epigenetic alterations that occur in HSCs or HSC niche compromise HSC function, resulting in MDS; therefore, MDS HSCs are likely the ideal targets for MDS treatment. Recent encouraging advances—capturing a molecular portrait of the whole genome of MDS CD34⁺ cells, including identifying altered signaling pathways and altered microRNAs—have improved our understanding of MDS pathogenesis and provided novel potential clinical targets for MDS. Here, I will briefly review the characteristics of MDS HSCs and discuss the therapeutic promise of targeting MDS HSCs.     

基于造血干细胞衰老及造血微环境损伤探讨骨髓抑制机制的研究进展

恶性肿瘤以其高发病率和高致死率严重威胁着人类的健康。化疗是临床治疗恶性肿瘤的有效手段,但常因并发骨髓抑制等副作用而限制其应用效果。骨髓抑制主要分为急性骨髓抑制和长期骨髓损伤两类,急性骨髓抑制主要是骨髓细胞凋亡所致,运用造血生长因子可以迅速得到缓解;但长期骨髓损伤临床缺乏有效的干预方案,严重影响化疗的连续性和治疗效果。因此,本文主要针对长期骨髓损伤,从骨髓造血干细胞衰老及造血微环境损伤两方面入手,查阅该领域近年来相关文献,综述其作用机制和相关通路靶点,为肿瘤基础研究提供文献支持,并为骨髓抑制新药开发提供新的思路。     

铁过载对造血干细胞移植的影响

铁过载与造血干细胞移植（HSCT）预后关系密切。移植前铁过载可能增加患者HSCT后发生感染、肝静脉闭塞病（VOD）及肝损伤的风险,降低患者移植后总生存（OS）率、无事件生存（EFS）率。移植后铁过载可能降低患者的OS率。祛铁治疗有可能使HSCT患者获益。     

Paraproteinemia after hematopoietic stem cell transplantation

The frequency and clinical significance of paraproteinemia in patients receiving hematopoietic stem cell (HSC) transplants were assessed. Of 66 patients with hematologic malignancies, excluding multiple myeloma who received an allogeneic or autologous HSC transplant, paraproteins were detected in 12 patients using immunoelectrophoresis. None of the patients showed paraproteinemia before HSC transplantation. The class of paraproteins most commonly seen was IgG. In 9 of these 12 patients (75%), a paraprotein was detected continuously after HSC transplantation for an average duration of 464 days, while others demonstrated a transient appearance of the protein. Paraproteinemia after HSC transplantation was not related to the stem cell source, (allograft vs. autograft), age, gender, viral infection and graft-vs.-host disease (GVHD). None of the patients developed plasma cell dyscrasia after the appearance of the paraprotein, while 1 patient developed secondary acute lymphoblastic leukemia. These findings indicate that paraproteinemia after HSC transplantation may be caused by an aberrant immune reconstitution after both allogeneic and autologous HSC transplantation. A long-term follow-up of patients with paraproteinemia after HSC transplantation is needed to confirm this finding in a larger series of patients.     

造血干细胞移植研究和应用的回顾与展望

 结合第54届美国血液学会年会教育论坛的相关内容着重复习造血干细胞移植（HSCT）领域的一些进展。在论述人类白细胞抗原（HLA）配型技术和供者来源扩展的基础上,分别简述了各类HSCT（HLA-相合同胞供者移植、脐带血移植、无关供者移植、 HLA-单倍型相合的移植）疗效的提升,也回顾了疾病种类和病期对HSCT结局的影响;结合自身的理解与经验,强调确保供者安全的注意事项与意义。     

Pre-transplant marital status and hematopoietic cell transplantation outcomes

BackgroundEvidence about the impact of marital status before hematopoietic cell transplantation (hct) on outcomes after hct is conflicting.MethodsWe identified patients 40 years of age and older within the Center for International Blood and Marrow Transplant Research registry who underwent hct between January 2008 and December 2015. Marital status before hct was declared as one of: married or living with a partner, single (never married), separated or divorced, and widowed. We performed a multivariable analysis to determine the association of marital status with outcomes after hct.ResultsWe identified 10,226 allogeneic and 5714 autologous hct cases with, respectively, a median follow-up of 37 months (range: 1–102 months) and 40 months (range: 1–106 months). No association between marital status and overall survival was observed in either the allogeneic (p = 0.58) or autologous (p = 0.17) setting. However, marital status was associated with grades 2–4 acute graft-versus-host disease (gvhd), p < 0.001, and chronic gvhd, p = 0.04. The risk of grades 2–4 acute gvhd was increased in separated compared with married patients [hazard ratio (hr): 1.13; 95% confidence interval (ci): 1.03 to 1.24], and single patients had a reduced risk of grades 2–4 acute gvhd (hr: 0.87; 95% ci: 0.77 to 0.98). The risk of chronic gvhd was lower in widowed compared with married patients (hr: 0.82; 95% ci: 0.67 to 0.99).ConclusionsOverall survival after hct is not influenced by marital status, but associations were evident between marital status and grades 2–4 acute and chronic gvhd. To better appreciate the effects of marital status and social support, future research should consider using validated scales to measure social support and patient and caregiver reports of caregiver commitment, and to assess health-related quality of life together with health care utilization.