自身免疫性血细胞减少症免疫发病机制、临床特征及利妥昔单抗疗效的研究（可编辑）

自身免疫性血细胞减少症免疫发病机制、临床特征及利妥昔单抗疗效的研究（可编辑） 自身免疫性血细胞减少症免疫发病机制、临床特征及利 妥昔单抗疗效的研究 天津医科大学 博士学位论文 自身免疫性血细胞减少症免疫发病机制、临床特征及利妥昔单 抗疗效研究 姓名:刘鸿 申请学位级别:博士 专业:临床医学;内科学血液病 指导教师:邵宗鸿 2012-05 天津医科大学博士学位论文 中文摘要 自身免疫性血细胞减少症是血液系统疾病中的一大类疾病，包括自身免疫 性溶血性贫血 成熟红细胞抗体介导 、免疫性血小板减少性紫癜 血小板抗体 、Evans综合征 成熟红细胞抗体和血小板抗体均有 、免疫相关性全介导 血 细胞减少症 未成熟血细胞抗体 等。长期以来对该类疾病发病机制的认识并 不清楚，仅局限于是由自身抗体介导的血细胞破坏所致，至于抗体产生的原因 和病理机制，则缺乏系统的认识。由于病理机制的研究没有大的进步，治疗效 果也并不满意。多年来该病治疗以肾上腺皮质激素为主，复发率高 多在80, 以上 。也有部分患者如冷抗体型自身免疫性溶血性贫血激素疗效差，缺乏更好 的治疗方法，患者常迁延不愈。本文探讨自身免疫性溶血性贫血 AIHA 患者 的免疫发病机制、难治及复发患者的新型免疫抑制剂利妥昔单抗的疗效及安全 性。另外免疫相关性全血细胞减少症是由本课题组提出，多年来致力于该病的 本文对该病的临床及实验室特征做出总结。通过对以发病及病理机制研究， 上 内容的研究，能对该类疾病的发生发展有更系统清楚的认识，提高治疗效果， 造福患者。 内 容 第一部分目的研究自身免疫性溶血性贫血 AIHA 的免疫发病机制。方法应 用流式细胞仪检测54例AIHA患者的免疫指标，其中B淋巴细胞方面，检测项目包 血发作组、缓解组，与正常对照组进行比较，进行三组之间的两两比较和统 计 学分析。应用SPSSl3(0统计软件进行分析，计量数据以均数?标准差的形式表 示，正态分布数据两组间比较以t检验，三组间比较以单因素方差分析;非正态 天津医科大学博士学位论文 结果1(AIHA患者中，B淋巴细胞虽PCDl9+细胞:溶血发作组较正常组明显增高， 正常对照组 P 0(033，P O(05 ;在患病组中，缓解组与溶血发作组比较， 妥昔单抗 治疗组:CDl9+:美罗华治疗组低于非美罗华治疗组 P 0(017， 的效应T细胞，即CD25+弄IHLA-DR+的T细胞，患病组与正常对照相比，差异无 统计学意义;但在T辅助细胞中检测Thl，Th2亚群时，发现患者的Thl细胞在 而Thl细胞在溶血发作组和缓解组差异无统计学意义 P O(05 ;而在Th2细胞， 随着免疫抑制剂的使用，Th2细胞降至正常，疾病也处于缓解状态。3(树突状细 胞 DC 检OH,0 低于缓解组 P O(011，P O(05 ，缓解组与正常对照组对比差异无统计学意义， 胞密切相关，在溶血发作状态，CDIg+、CDSCDl9+两群淋巴细胞比值明显增高， 提示存在B淋巴细胞增殖;在利妥昔单抗治疗组 美罗华组 CDl9f 一淋巴细胞低 TT 天津医科大学博士学位论文 于非美罗华治疗组，CD5+CDl9+细胞美罗华治疗组与非美罗华治疗组差异无统计 学意义，提示美罗华免疫抑制作用明显较强;T淋巴细胞的调控尤其是Th2细胞 增殖与AIHA的发病亦密切相关，溶血发作时Thl、Th2比值均明显增高;缓解 时Th2比值明显降低，Thl无明显变化;DC亚群与A工HA发病也有关系，mDC、 pDC在发病时比值均明显减低，经免疫抑制剂治疗后，mDC结果趋于正常，pDC 比值仍明显降低，提示溶血发作时可能存在DC的外周淋巴组织迁移。 第二部分:目的进行病例分析，探讨新型免疫抑制剂利妥昔单抗联合环磷酰胺 治疗难治性自身免疫性溶血性贫血疗效，并进行长期随访，对其及长期疗效及 安全性做一评价。病例自身免疫性溶血性贫血7例 其中Evans综合征1例 ， 次;环磷酰胺:lg,次，每10天1次，连用2―7次;同时加用静注免疫球蛋白 ,次，1次,周，利妥昔单抗之后1天使用。结果所有患者3个月时均有59 效 7,7 ， 完全缓解 CR 率占6,7，部分缓解占1,7，平均随访27月，12个月复查时所 有完全缓解患者均未见复发，部分缓解患者血红蛋白正常，仅间接胆红素升高， 网织红细胞升高;24个月复查时2例患者出现间接胆红素升高，网织红细胞升 高，l例患者单独加用利妥昔单抗强化治疗后再达CR，1例未加用其他强化治疗， 仍用环孢菌素A维持治疗。36个月时该未强化患者复发，经加用上述利妥昔单 抗+环磷酰胺方案治疗3疗程再达部分缓解。所有患者对该治疗耐受性好，不良 反应轻微。结论利妥昔单抗联合CTX用于治疗难治性自身免疫性溶血性贫血， 疗效较前显著，未见严重不良反应，但停药12-24个月后部分患者有复发趋势， 再用利妥昔单抗进行强化治疗仍有效。 第三部分:目的总结157例免疫相关性血细胞减少症 IRP 患者的临床特征。 方法回顾性分析天津医科大学总医院血液科课题组2006年1月至2010年7月 诊治的157例骨髓单个核细胞膜抗体试验阳性的IRP患者的基本情况、发病诱 因、临床表现、血象及骨髓象、自身抗体类型及免疫治疗效果。结果44(6, 70,157 IRP患者发病前有感染、过敏、妊娠及装修等诱因。全血细胞减少者 TIT 天津医科大学博士学位论文 重度均可见，贫血类型以大细胞贫血为主，占61(3, 95,155 ，其次为正细胞贫 减少[91(7, 144,157 ]，可见血小板轻、中、重度减少，出血表现以皮肤黏 膜出血为主。髂骨骨髓增生多为活跃和明显活跃68(8, 108,157 ，少数 增生减 大部分患者[85(7, 102,119 1胸骨骨髓为增生活跃或明显活跃。巨核细胞数 多合并血小板形成不良。流式细胞仪检测的骨髓单个核细胞膜抗体试验皆阳 性，检出不同细胞群的自身抗体，各种类型和组合方式均可见，提示自身抗体 性。共有56(7, 89,157 的患者合并轻微溶血，但不符合任何的多克隆 种类的溶 血性疾病的诊断标准:57(9, 91,157 患者同时出现其它自身免疫指标异常， 低，给予免疫抑制和促造血治疗后，3年有效率为86(8, 33,38 。结论IRP是 一类获得性自身抗体介导的骨髓细胞破坏或抑制性疾病，自身抗体为多克隆性， 约半数患者有感染或过敏等诱因，主要表现为网织红细胞或 和 中性粒细胞 比例不低的两系或三系血细胞减少，多数患者至少一个部位骨髓为增生活 跃， 常有轻微溶血迹象但溶血试验均为阴性，常合并其他自身免疫指标异常，如补 体降低，抗核抗体阳性等，对免疫抑制和促造血治疗反应好。 全文结论AIHA的发病与B淋巴细胞及CD5+B淋巴细胞密切相关，受到Th细胞 TH2细胞可能起主要作用; 调控， 溶血发作时mDC、pDC均减少，可能与溶血发 作时的外周淋巴组织转移有关。利妥昔单抗治疗难治复发AIHA效果较好，但1 年后可有复发;IRP是一类获得性自身抗体介导的骨髓细胞破坏或抑制性疾病， 自身抗体为多克隆性，约半数患者有感染或过敏等诱因，主要表现为网织红细 胞或 和 中性粒细胞比例不低的两系或三系血细胞减少，多数患者至少一个 TV 天津医科大学博士学位论文 部位骨髓为增生活跃，常有轻微溶血迹象但溶血试验均为阴性，常合并其他自 身免疫指标异常，如补体降低，抗核抗体阳性等，对免疫抑制和促造血治疗反 应好。 关键词 发病机制;利妥昔单抗;溶血;血细胞减少;自身免疫性 V 天津医科大学博士学位论文 Abstract of was to the 1 This Section designedinvestigatepathogenesis Objectivestudy Autoimmune HemolyticAnemia AIHA ( weredetectedflow blood indexes Methods:The by cytometryusing following weretheamountofCD19+and from54 with patientsAIHA(They samples amountofCD4+andCD8+T 19+B CD5+CD cells，the lymphocytic lymphocytic Thl andTh2T amountofNK amountof helpercells CD4+cells ， cells，the cell，the effective HLA―DR+T theamountofT cellsandT cells CD25+andcells ， regular weredividedinto theamountofmDCand dendriticcells(Patients hemolytic pDC indexesfromeach were attack andremission statistically group group(The group Measurementdataweredescribedasmean+standard and analyzedcompared( SPSSl3(0statistics twonormal software，wecompared deviation(Applying on basedon abnormaldistributiondatabased distributiondata t-test， of three test Kruskal―Wallistest ，weanalyzedgroupsbyanalysis non―parametric wasdefinedasP 0(05( significance variance one―wayANOVA (Statistical Results1(Bcells:TheamountofB lymphocytic attack was thanthatfromnormalcontrol significantlyhigher hemolyticgroup 19+B from attack werein P 0(000， 0(01 (CD5+CDcells hemolytic group normal CD5+CDl9+ excessofthosefrom from attack Bcellsfromremissionwere lowerthan group significantly hemolytic 19+, CD19+from 1 (TheratioofCD5+CD group P 0(000，P O(000，P O(O lowerthanfrom attack 1 (The remissionwas hemolyticgroup P 0(004，P O(O group 19+cells ofCD5+CD19+,CD19+ amountofCD1 andtheratio 9+cells，CD5+CD healthcontrol 1， werelowerthanfrom alsodividedthe ontreatmentwhether P 0(01，P 0(05 (We based patients using rituximab(TheamountofCD19+cellsfromrituximabtreatmentwaslower group 1 no difference thanthe 7，P 0(05 (Therewas counterpartgroup P 0(0 statistically ofCD5+CD19+cell betweenrituximabandthe quantity group counterpartgroup( VT 天津医科大学博士学位论文 rituximabwas thanthe 19+,CD19+from TheratioofCD5+CD group higher counterpartgroup P 0(032，P 0(05 ( differencesoftheamountof wereno CD4+cells， 2(Tcells:There significant HLA―DR+Tcellsbetween and and patients CD8+cells，NKcells，CD25+cells wasnot differentbetweenthe controls(TheratioofCD4,CD8 significantly healthy Thlcellsfrom attack either(Theamountof two hemolyticgroup P 0(002， groups both thannormalcontr01( P 0(05 andremission higher group P 0(021，P 0(05 was ofThlcellsbetween no differencesoftheamount Therewas hemolytic significantly ofTh2cellsfrom andremission attack group P O(05 (Thepercentage group thancontrol P 0(000，P 0(0 attack was 1 (Meanwhile， the hemolyticgrouphigher waslowerthan attack ofTh2cellsfromremission hemolyticgroup group percentage thatafter 1 (Thisindicated P O(002，P 0(0 immunosuppressivetherapy， Th2 thediseasetendedintoremission( cellstendedtobecomenormalwhile amountofCD123+cellsfrom waslowerthan 3(Dendritriccells:The patients ofCDllc+cellsfrom attack control P 0(000，P 0(01 ，the hemolytic percentage wasno 11，P 0(05 (There waslowerthanremission statistically group P 0(0 group remissionandcontrol differenceofCD11c+cellsbetween group(The group attack andremission ofCDl23+cellsfromboth group hemolyticgroup percentages wasno werelowerthancontrol P O(000，P O(001 (There statisticallysignificance 123+cellsbetween attack andremission oftheamountofCD hemolyticgroup group。 c+,CD123+fromboth attack TheratioofCD11 hemolytic thancontr01(Therewasno 1 were andremission higher group P O(002，P 0(0 oftheratiobetween attack andremission differences hemolyticgroup significantly group( withactivationofB The ofAIHAisassociated Conclusions pathogenesis of 1 the attack，the cells CD5+CD9+ (Duringhemolytic percentages lymphocytic CD5+CD19+Bcells the CD19+Bcellsand substantiallyelevate， indicating 1 cellsfrom ofB cells(TheamountofCD lymphocytic 9+lymphocytic proliferation thanthe therewasno rituximabwaslower counterpartgroup，while group VlI 天津医科大学博士学位论文 ofCD5+CD19+cellsbetweenrituximabandthe significance statistically group thatrituximabexhibitsa inhibitioneffect counterpartgroup(thissuggests strong towardsimmune ofT Th2 system(Theregulationlymphocytic cells especially the ofAIHA(The alsorelatedto mechanism ofThland proliferation is percentages Th2cellsbothincreased Th2decreasedwhenremission attack，and duringhemolytic was whileThlshowednoalteration(The achieved ofAIHAisrelated pathogenesis cells toDCcells(The ofDC1 andDC2 bothdecreasedthe percentages during DC1 tendedtobecomenormalwhileDC2decreasedafter attack，yet indicatesthatDCs into (This maybe therapy immunosuppressive migrate tissuewhen attacks( peripherallymphoid hemolysis Section Tobetterassessthe and ofmonoclonal 2(0bjective efficacysafety anti―CD20 rituximabcombined withintreatmentof antibody cyclophosphamide andrecurrentautoimmune anemia( CasesThe refractory hemolytic study included7casesofautoimmune 1 caseofEvans hemolyticanemia including allofthemwere orrecurrent(Methodsrituximab: syndrome ，which refractory 375 mg,m2，onceweek，2-6 times; per times;Cyclophosphamide:1g,10days，2-7 combinedwith intravenous 1 after immunoglobulin IVIG 5g,week，givenday rituximabtreatment(ResultsAll7 showed patients goodresponses(6patients achieved 1 achieved completeremission CR andpatient partialremission PR ( occurred1 1 to0monthsafterthefirstdoseofrituximabandthemean Responses effectivetimeis 2(5 forthe is approximatelymonths(Averagefollow-uppatients around27months(Allremainedinremissionatthe12一month patients follow―up visits(Atthetimeof24一month showedelevatedindirectbilirubin visits，2 patients andincreased counts(One achievedCRafteradditional reticulocyte patient rituximab theother waitandwatchwithout additional therapy,andpatientjust thetimeof 36一month andwasretreatedwith3 visits，1 therapy(At patientrelapsed ofrituximaband reached PR(All toleratedthetreatment cycles eventually patients wellwith mildsideeffects(Conclusionsrituximabcombinedwithand only Vlll 天津医科大学博士学位论文 in is effectiveand safe recurrentautoim highly relativelypatients cyclophosphamide treatmentcanbe in with mune anemia(Additional given refractoryhemolytic 1-2 with after relapse years( patients To theclinicaland featuresofthe Section study laboratory patients 3(Objective Therisk immuno―related with factors， manifestations， pancytopenia IRP (Methods and marrow bloodcell counts，bonephenotypes，autoantibodies with werefollowed of157 IRPwere patients analyzed(Thenthey therapyresponse their outcomeandthe factors( see prognostic upfor 3-48 months，tolong―term and were tobethe ResultsThe infection，anaphylaxispregnancyhighlysuspected with ofIRP(Mostofthese were riskfactors 73(2, 115, 157 ， patients pancytopenia werewithanemiaand some thrombocytopenial8(5, 29 ,157 ]; patients themwereanemicwith them largeMCV，32(9, 51,155 of 61(3, 95,155 of normal 24,1 themwerewith wereanemicwith MCV;78(9, 157 of leukopenia， was themhad 57 of fever(Thrombocytopeniacommon[91(7, 23(6, 37,1 with serious was caseswere rare(68(8, 108,157 ofthese bleeding 144,157 ],but cellularitiesandincreased were nomalorincreasedbonemarrow normoblasts(They cell Facs tohave resultsofbonemarrowmononuclear allfound antibody positive noevidenceof clonal resultsofrutine testsand hemolysis malignant test，negative these had 18(5, 57 of C3decreased，and patients hematopoiesis(42(6, 67,1 wasadministeredto157IRP 29,157 C4 therapy decreased(Immunosuppressive rateat 36monthswas patients，Theresponse an multiclonal hnmuno―related acquired pancytopenia IRP is without conductedbonemarrowfailure syndrome，featuringpancytopeniareducing thanhalfofthe usedto of and patients reticulocytesneutrophilicgranulocytes(More few marrowhave at haveinfectionsor patients’bone higherproliferation allergies(A IX 天津医科大学博士学位论文 test(The mild hemolytic following leastone yetnegative site，withhemolysis aslowamotmtof with abnonnalitiesoften IRP,such complements， complicates to receive ANA(Those responses patientsmay good positive treatment( and hemopoiesisimproving withB AIHAis associated The of Conclusions closely lymphocytic pathogenesis cellsofB cells(Th2 cellsandCD5+B cells，theregulating lymphocytic lylnphocytic amountofmDCand in an pDC hemolysis(The importantpart cells，mayplay DC caused decrease attack，which peripheralmigration by duringhemolytic 1 later( sometimes has to AIHA Rituximab，butrelapseyear response Refractory good isan multMonal Imnmno(related acquired pancytopenia without failure conductedboneman'ow pancytopeniareducing syndrome，featuring halfofthe usedto than of and patients reticulocytesneutrophilicgranulocytes(More at marrowhave few haveinfectionsor higherproliferation patients’bone allergies(A test(The mild leastone negativehemolytic following site，withhemolysisyet lowamountof with as abnonnalitiesoften IRP,such complements， complicates receive to ANA(’Fhose responses patientsmay good positive treatment( and hemopoiesisimproving immune―related rituximab; pancytopenia; KeywordsPancytopenia; anemia hemolytic cyclophosphamide;autoimmune X 天津医科大学博士学位论文 缩略语,符号说明 AIHA autoimmuneanemia hemolytic 自身免疫性溶血性贫血 CsA 环孢菌素A cyclosporine IRP immuno-related 免疫相关性全血细胞减少 pancytopenia RC 网织红细胞 reticulocyte mean MCH 平均血红蛋白 含量 corpuscularhemoglobin MCHCmean hemoglobin 平均血红蛋白浓度 corpuscular concentration MCV meancellvolume 平均红细胞体积 MDS 骨髓增生异常综合征 myelodysplasticsyndrome Ct CDl23interleukin一30c receptor 白介素受体-3 DC cell Dendritic 树突状细胞 DI differenl:iationindex 分化指数 EP0 促红细胞生长素 erythropoietin FACS fluoresceneactivatedcell sorting 荧光激活细胞分类 FCM flow 流式细胞术 cytometry FITC fluorescein 异硫氰酸荧光素 isothiocyanate G(CSF factor 粒系集落刺激因子 granulocytecolony―stimulating GM。CFU 粒一巨噬细胞集落形成单 granulocyte―macrophagecolonyforming unit 位 IPSS international prognostic 国际预后积分系统 scoringsystem LBR laminB 核纤层蛋白受 体 receptor LDH lactate 乳酸脱氢酶 dehydrogenase LSC leukemiastemcell 白血病干细胞 ITP Immune 免疫性血小板减少性紫癜 thrombocytopenicpurpura PAS acid―schiff 过碘酸一雪夫反应 periodic PE phycoerythrin 藻红蛋白 PerCP peridininphyllochlorin 多甲藻素一叶绿素蛋白 PI iodide 碘化丙啶 propidium PLT platelet 血小板 XIII 磷脂酰丝氨酸 PS phosphatid ，lserine 难治性贫血 anemia RA refractory blasts 难治性血细胞 减少伴原 anemiawitheXCeSS RAEBrefractory 始细胞增多 sideroblasts anemiawith 难治性贫血伴 环形铁粒 RARS ringed refractory 幼细胞增多 红细胞 cell RBC redblood with 伴有多系发育 异常的难 RCMD multilineage refractorycytopenia 治性血细胞减少 dysplasia with 伴有单系发育异常的难 unilineage RCUD refractorycytopenia 治性血细胞减少 dysplasia d