【作者】 杨惊；

【导师】 赵永强； 王书杰；

【作者基本信息】 中国协和医科大学 ， 血液病学， 2010， 博士

【摘要】 目的：评价成人ITP患者TPO水平与激素治疗及预后的关系,T、B淋巴细胞功能异常与病情活动的相关性。方法：1、收集2008年7月至2009年7月就诊于北京协和医院的血小板减少症患者的临床资料及外周血标本；2、酶联免疫分析法(ElISA)测定原发性ITP组(PITP)、系统性红斑狼疮相关ITP组(SLE-ITP)、正常对照组3个实验组的人血浆血小板生成素(TPO)水平并进行比较；3、流式微球分析(CBA)法测定血浆中Th1/Th2/Th17细胞因子(IL-2、IL-4、IL-6、IL-10、TNF、IFN-γ、IL-17A)水平；4、改良间接MAIPA法测定血浆中抗血小板GPI和GPIIb特异性抗体；5、B细胞酶联免疫斑点检测技术(ELISPOT)测定外周血单个核细胞中抗GPIIb/IIIa特异性效应B细胞和记忆B细胞的频数；6、CBA法测定单个核细胞培养上清的Th1/Th2/Th17细胞因子水平。结果：1、就诊的血小板减少症患者共252例,其中PITP132例,继发性ITP 54例。PITP共132例中男性51例、女性81例,中位发病年龄为38岁,有出血表现者为77例,发病时的血小板计数的中位值为25×109／L,骨髓巨核细胞计数中位值为186个／片。抗核抗体(ANA)阳性者33例(25%),LA阳性4例,β2GP1阳性者3例。2010-4电话随访88例,其中激素治疗可获得完全缓解者53例(60%),部分有效者21例(24%),无效者14例(16%)。ANA阳性组激素治疗后复发及治疗无效的比例均显著高于ANA阴性组(P<0.05)。2、血浆TPO水平,PITP组(256.20±846.20pg/ml)和SLE-ITP组(195.69±383.15pg/ml)TPO值均高于正常对照组(22.83±32.46 pg/ml),有显著性差异(P<0.01),TPO水平与血小板计数呈负相关(r=-0.488,P值<0.01),与骨髓巨核细胞计数无明确相关性(r==-0.160,P值=0.116)。激素治疗持续CR (CCR)组12例中1例TPO增高,复发组则有25%(8／32)TPO水平增高,而激素无反应组则有33.3%(4／12)TPO水平增高。三组间TPO值及TPO增高者比例均无显著差异。3、PITP组与正常对照组相比,IL-2,IL-4, IL-6, IL-10, TNF, IFN-y和IL-17A水平均无显著性差异。SLE-ITP组与PITP组比较,SLE-ITP组IL-10水平显著高于PITP组,血小板计数和IL-2. IL-4. IL-6. TNF. INF-y和IL-17A水平与PITP组相比均无显著差异。血小板计数与IL-17A(r=0.149,P=0.038)和IFN-y(r=0.142,P=0.046)呈正相关,但相关性不强；IL-17A与IL-2. IL-4. IL-6. IL-10. TNF. IFN-y均显著正相关(r=0.647,0.892,0.879,0.644,0.253,0.921,P<0.01)。4、93例PITP和26例SLE-ITP患者进行改良MAIPA检测。PITP组MAIPA-GpI阳性率为22.6%,MAIPA-GpIIb为23.7%,SLE-ITP组MAIPA-GpI为46.2%,MAIPA-GpIIb 57.7%, PITP缓解组8例中仅有MAIPA-GpIIb 1例阳性。PITP组MAIPA总阳性率高于PITP缓解组而低于SLE-ITP组,p<0.01。MAIPA阳性组患者IL-6浓度与IL-10浓度显著高于阴性组。5. ELISPOT法测定抗GpIIb/IIIa特异性效应B细胞频数,在PITP组(n=70)、SLE-ITP组(n=16)和激素治疗CR组(n=10)分别为8.18±27.22/106PBMC, 7.90±20.81/106PBMC,和3.50±4.47/106PBMC,三组之间无显著性差异；正常对照组(N=10)的频数为1±1.13/106PBMC,显著低于PITP组(P=0.03)。加入含PWM+SAC B细胞刺激培养组产生IgG的B细胞数为288.26±355.43／105PBMC,未刺激组为28.53±54.82/105PBMC。PWM+SAC刺激组产生总Ig的B细胞数显著高于未刺激组。PITP活动组组特异性GPIIb-IIIa记忆B细胞实际频数(27.35±30.13%)显著高于SLE-ITP(11.49±16.19%)(P<0.01),与激素治疗CR组(28.47±29.60%)无显著差异。6、CBA法共测定34例患者单个核细胞培养上清Th1/Th2/Th17细胞因子,PWM+SAC刺激组IL-4、IL-6、IL10、TNF、IL-17A值均显著高于未刺激组。PWM+SAC刺激组IL-4、IL-6、IL10、TNF、IL-17A浓度与总记忆B细胞频数呈显著正相关(P<0.01),IL-4、IL-6、IL10浓度与特异性记忆B细胞频数呈显著正相关(P<0.05)。结论：1、血小板减少症以原发性ITP最多见,继发性ITP的病因多为自身免疫性疾病(尤其SLE)。部分原发性ITP患者可有ANA阳性,ANA阳性者激素疗效较差。2、成人慢性原发性ITP患者的TPO水平较正常组显著升高,血小板数目越低者TPO水平越高,激素无效者较激素CCR者TPO水平较高但差异不显著,TPO水平与巨核细胞增生程度之间的关系暂不明确。3、成人慢性原发性ITP患者的Thl、Th2细胞因子分泌水平与正常对照相比并无显著差异,Th2细胞在血小板特异性抗体产生中可能起着重要的作用,Th17‘细胞可能在抗GPI抗体产生中起到了一定作用。4、PITP与SLE-ITP组患者外周血单个核细胞中特异性GPIIb-IIIa效应B细胞的频数均明显高于正常对照,Elispot方法检测特异性GPIIb-IIIa效应B细胞不受ITP患者是否进行激素治疗的影响,不论其是否缓解均可进行检测,但无法区分PITP和SLE-ITP。5、Th2细胞在ITP记忆B细胞体外培养活化中有关键作用,激素治疗并不能清除PITP患者外周血中的记忆B细胞。6、PITP组特异性GPIIb-IIIa记忆B细胞实际频数显著高于SLE-ITP,特异性记忆B细胞实际频数可能有助于PITP和SLE-ITP的鉴别。更多还原

【Abstract】 Objective:To evaluate the relationship between the plasma TPO level and the outcome of treatment of steroids and correlation of abnormalities of T, B cell function with the activities of the illness in the adult ITP patients.Methods:1、Collected clinical data and blood samples of the adult ITP patients in Peking Union Medical College Hospital from July 2008 to July 2009.2、Three groups haved been enrolled to the study:primary immune thrombocytopenia (PITP)、ITP secondary to Systemic lupus erythematosus (SLE-ITP) and normal control.Plasma thrombopoietin (TPO) levels were measured using Enzyme-linked immunosorbent assay (Elisa) and compared among the three groups.3、Cytometric bead array (CBA) was used to measure the plasma concentration of seven Thl/Th2/Thl7-associated cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, Tumor necrosis factor (TNF), interferon-gamma (IFN-γ)).4、Anti-GPⅡb/Ⅲa and anti-GPIb/IX autoantibodis were detected by modified monoclonal antibody immobilization of platelet antigens assay (MAIPA).5、B cell enzyme-linked immunosorbent spot (ELISPOT) assay were used to detect human effective and memory B cells producing anti-GPⅡb/Ⅲa antibodies in human peripheral blood mononuclear cells(PBMCs).6、Concentrations of Th1/Th2/Th17-associated cytokines in the supernatant of mononuclear cells culture were determined by Cytometric bead array (CBA).Results:1.252 thrombocytopenic patients, including 132 PITP and 54 secondary ITP, were enrolled into the study. Among 132 case of PITP, female-to-male ratio was 81:51, with a median age of 38 years old, median platelet count of 25 x 109/L, median megakaryocyte number in bone marrow was 186 per smear. Out of 132 cases,33 (25%) were ANA positive,4 cases LA positive,3 casesβ2GP1 positive.88 patients who were treated by steroids have received telephone inquisition in April 2010. Among them,53 (60%) achieved complete remission (CR),14 cases (16%) had no response. In comparison with the ANA negative patients, ANA positive patients had more steroid non-responders and higher recrudescence (P<0.05)2. PITP group (256.20±846.20pg/ml) and SLE-ITP group (195.69±383.15 pg/ml) had markedly elevated TPO levels in comparison with the control (22.83±32.46 pg/ml) (P< 0.01).There was a significant negative correlation between their plasma TPO concentrations and platelet counts (r=-0.488, P<0.01), no significant correlation was found between plasma TPO concentrations and megakaryocyte count (r=-0.160, P=0.116).There was no significant difference in TPO levels between steroid CR and steroid non-responders group.3. The levels of IL-2, IL-4, IL-6, IL-10, TNF, IFN-y and IL-17A had no significant difference between PITP and normal control. The IL-10 level in SLE-ITP was significant higher than that in PITP. There was a weakly positive correlation between platelet counts and the level of IL-17A (r=0.149, P=0.038),and also between patelet and IFN-y (r=0.142, P=0.046). The level of IL-17A was positively correlative to level of IL-2、IL-4、IL-6、IL-10、TNF or IFN-y (r=0.647,0.892,0.879,0.644,0.253,0.921,P<0.01)4. The positive rates of antibodies against platelet GPIb and GPIIb in PITP group were 22.6% and 23.7%, whereas they were 46.2% and 57.7% in SLE-ITP group, respecitively. In 8 PITP-CR patients, one patient had positive MAIPA-GpIIb only. In comparison with patients with negative MAIPA detection, those who had positive detection had higher levels of IL-6 (P<0.05) and IL-10 (P<0.01)5. The frequencies of GPIIb-IIIa-reactive B cells determined by ELI SPOT were 8.18±27.22/106PBMC in PITP group (n=70),7.90±20.81/106PBMC in SLE-ITP group (n-16) and 3.50±4.47/106 PBMC in PITP—CR group, respectively. There were no significant differences among three groups. The frequency in PITP-active group was significantly higher than that in control group (1±1.13 per 106PBMC,n=10), P value was 0.03. The total reactive B cell frequencies in PWM+SAC stimulated group was significantly higher than no stimulate group (288.26±355.43/105 PBMC vs 28.53±54.82/105 PBMC).The actual gpIIb-IIIa-reactive memory B cells frequencies in PITP-active group (27.35±30.13%) were significantly higher than SLE-ITP group (11.49±16.19%), but no significantly difference between PITP-active and PITP-CR groups.6. The levels of IL-4、IL-6、IL10、TNF and IL-17A in cell culture supernatant in PWM+SAC stimulated group were significantly higher than those in no stimulate group. There was a significant positive correlation between level of IL-4、IL-6、IL10、TNF or IL-17A in cell culture supernatant in PWM+SAC stimulated group and the number of total reactive B cells (P<0.01), also a significant positive correlation between levels of IL-4、IL-6、IL10 and actual gpIIb-IIIa-reactive memory B cells frequencies.Conclusions:1. Most cases of thrombocytopenia were primary ITP. Secondary ITP comprised heterogeneous disorders which were usually associated with systemic autoimmune diseases, especially SLE. In some PITP patients, ANA were positive, which might predict steroid refractory.2. The level of TPO in adult chronic PITP patients was significantly higher than that in healthy controls, and lower platelet count was associated with higher TPO level. The relationship between megakaryocyte hyperplasia, outcome of steroid therapy and TPO levels were not clear.3. No significant differences in the concentration of Th1/Th2/Th17 cytokines were observed between patients with PITP and the control.Th2 cells may play an important role in the production of platelet autoantibody, Th17 may be associated with the production of antibodies against platelet GPIb.4. The frequencies of GPIIb-IIIa-reactive B cell in peripheral blood from PITP and SLE-ITP were significantly higher than that from normal control. Detection of GPIIb-IIIa-reactive B cell by ELISPOT was not affected by steroid treatment, but may not be helpful in distinguishing PITP and SLE-ITP.5. Th2 cells played a key role in the activation of ITP memory B cells in vitro, steroid therapy could not clear the memory B cells out of peripheral blood of PITP patients. 6. The actual gpIIb-IIIa-reactive memory B cells frequencies in PITP-active group were significantly higher than SLE-ITP group, the actual gpIIb-IIIa-reactive memory B cells frequencies may be helpful in distinguishing PITP and SLE-ITP.更多还原