【作者】 李建琴；

【导师】 王兆钺；

【作者基本信息】 苏州大学 ， 内科学， 2013， 博士

【摘要】 免疫性血小板减少性紫癜（immune thrombocytopenia，ITP）是一种以血小板免疫性破坏为特征的自身免疫性疾病。临床上分为慢性ITP与急性ITP，慢性ITP多发病于20~50岁之间，女性发病率较男性高2~3倍，绝大多数慢性ITP患者缺乏临床表现或明确的病因。急性ITP是儿童比较常见的出血性疾病，发病年龄以2～5岁之间为多，病程一般4～6周，多数患儿病情可自行缓解，痊愈后很少复发，多在病毒感染或预防接种后发生，成人少见，无性别差异，通常在冬春季节、病毒感染高峰期发病较多。根据临床经验，急性ITP大多可自然缓解，而慢性ITP起病隐匿，病因复杂。越来越多的证据表明，ITP患者除体液免疫功能紊乱，产生自身抗血小板抗体外，还存在T细胞亚群的失衡，如Th1/Th2平衡失调，并以Th1异常表达占优势，另外有研究表明，Treg及Th17细胞数目均发生异常。目前临床对于ITP的治疗主要是通过三种途径来达到血小板数目的增加：（1）通过抑制血小板的清除，比如脾切除术；（2）通过抑制或者纠正异常的免疫反应，比如皮质甾醇类药物或者rituximab；（3）通过增加血小板的生成，比如TPO-RAs。但是对于部分慢性ITP患者的治疗效果却不显著，临床比较棘手。有报道指出，短程大剂量地塞米松冲击疗法对慢性ITP患者有较好的治疗效果，但是机制不明。本研究试图研究地塞米松对ITP患者的治疗效果并阐明其作用机制。本文首先对ITP患儿外周血中的T细胞亚群，包括Th1、Th2、Treg及Th17细胞进行流式分析，并对IL-2、IFN-γ、IL-4、IL-10、TGFβ及IL-17等细胞因子进行ELISA测定，对T-bet、GATA-3、Foxp3及RORγt进行实时定量PCR分析，初步探讨ITP患儿中T细胞亚群的改变。再进一步对慢性ITP患者进行短程大剂量地塞米松冲击疗法，观察其治疗效果并阐述其作用机制。我们对30例正常对照和30例ITP患儿进行Th1、Th2、Treg、Th17的流式分析，结果表明，Th1细胞在正常对照与ITP患儿中没有显著变化，而Th2细胞在ITP患儿中的表达显著降低，ITP患儿中Treg细胞数目减少而Th17细胞数量增加。用ELISA方法分别检测各细胞因子含量，结果发现，Th1的细胞因子（IL-2及IFN-γ）在ITP患儿外周血中含量明显升高，而Th2的细胞因子（IL-4和IL-10）含量明显降低，TGFβ及IL-17的表达与Treg及Th17细胞含量结果一致，分别为降低和升高。 Realtime PCR结果显示ITP患者外周血PBMC中T-bet的表达没有显著变化，但是GATA-3及Foxp3的表达在ITP患者中显著降低，而RORγt的表达明显升高。我们对10名ITP患者进行短程大剂量地塞米松冲击治疗，结果表明70%的患者在治疗后血小板数量明显增加，可达到正常水平。我们对T细胞亚群及转录因子进行了分析，结果表明，地塞米松治疗的患者外周血Th2细胞及Treg细胞较未治疗组有明显的升高，Th17细胞有显著的降低，同时我们发现转录因子表达改变趋势与相关T细胞亚群相同。为了进一步揭示用地塞米松纠正T细胞亚群失衡来治疗ITP的机制，我们纯化分离正常的T细胞，地塞米松体外处理。结果表明地塞米松可以抑制RORγt的表达，但是促进GATA-3及Foxp3的表达。这些结果表明地塞米松是通过调节转录因子的表达水平从而影响T细胞亚群的分化来纠正ITP患者中T细胞亚群异常的状态，从而达到治疗ITP的目的。综上所述，我们首次对儿童ITP的T细胞亚群的含量、细胞因子及转录因子的表达进行了分析，对异常T细胞亚群参与ITP的发病提供了更多的证据，并对大剂量地塞米松治疗ITP患者的机制进行了初步探讨，这些研究结果为揭示ITP的发病机制并为治疗ITP提供了重要信息。更多还原

【Abstract】 Immune thrombocytopenia (ITP) is a kind of platelet immunological failure featuresof autoimmune disease. Clinically, ITP is divided into acute and chronic ITP. Chronic ITPdisease is mainly in20-50years old adult people. Female incidence rate is2-3times morethan that of male. Most chronic ITP patients lack clinical symptoms and definite reasons.Acute ITP is mainly occurred in children about2-5years old after virous infection andvaccination and there is no gender difference. The general course of acute ITP is four to sixweeks and the majority of children with the disease can relieve itself, rarely relapse afterrecovery. Acute ITP usually happens in spring/winter and the fastigium of virous infection.According to clinical experience, acute ITP is mostly spontaneous remission. However thecause of chronic ITP is complex. More and more evidence shows that, other than humoralimmune function disorders, imbalance of Th1/Th2is happened in ITP patients and Th1type reaction is dominant. In addition, it is reported that the number of Treg and Th17cellswere abnormal in ITP patients.Recent progress of ITP treatment enables us to use three distinct approaches toincrease platelet mass:(1) by suppression of platelet clearance, e.g. splenectomy,(2) bysuppression or modification of abnormal immune responses, e.g. corticosteroids orrituximab, and (3) by stimulation of platelet production, e.g. TPO-RAs. But for somepatients with chronic ITP, the treatment effect is not significant.There are reports of short-range high concentrations of dexamethasone has a goodtherapeutic effect for patients with chronic ITP, but the mechanism is unknown. Thisresearch attempts to study the therapeutic effect of dexamethasone in patients with ITP andto elucidate its mechanism of action. In this paper, we first analyzed the T cell subsets in the peripheral blood of ITP patients using FCM including Th1, Th2, Treg and Th17cells.Meanwhile, IL2, IFN-gama, IL4, IL10, TGFbeta and IL17cytokines were assessed byELISA; mRNA of T-bet, GATA-3, Foxp3and ROR γ t were checked using real timefluorescent quantitative PCR. Furthermore, we evaluated the therapeutic effect of shortcourse-high concentration of dexamethasone in the treatment of patients with chronic ITPand studied the mechanism of dexamethasone in treatment of ITP.We analyzed30normal people and30ITP patients. Our results showed that thecontent of Th1cells did not change significantly in normal subjects and in patients withITP, and the content of Th2cells in patients with ITP was significantly lower. At the sametime, we analyzed another two T cells subsets Treg and Th17. The results showed that thenumber of Treg cells was reduced and Th17cells increased in patients with ITP. ELISAresults showd that Th1type cytokines (IL2and IFN-gama) were increased in the peripheralblood of patients with ITP, and Th2type cytokines (IL4and IL10) was significantlydown-regulated.As the change of Treg cells and Th17cells, TGFbeta and IL17were also decreasedand increased respectively in the peripheral blood of patients with ITP. Realtime PCRresults showed there were no significant changes of the expression of T-bet in peripheralblood of patients with ITP, but the expression of GATA-3and Foxp3were significantlylower in patients with ITP, and the expression of ROR γ t is higher than normal people.Subsequently, we treated10ITP patients with high concentration of dexamethasone. Thetreatment results showed that70%of the patients after treatment significantly increased thenumber of platelets. At the same time, T cell subsets and transcription factors wereanalyzed. The results show that, Th2cells and Treg cells in peripheral blood of patientstreated with dexamethasone compared to untreated group significantly increased, and Th17cells were significantly decreased. At the same time, we found that expression oftranscription factor is changed as T cells subsets. In order to further reveal the mechanismof dexamethasone in changing the T cell subsets in ITP, we isolated the T cells from normal people and treated with dexamethasone. The results show that dexamethasone caninhibit the expression of ROR γ T, but promotes the expression of GATA-3andFoxp3.These results suggest that dexamethasone can correct the T cell subsets in patientswith ITP by regulating the expression level of transcription factors.In summary, we for thefirst time analyzed the content of T cells subsets, cytokines and transcription factors in ITPpatients, providing more evidence for abnormal T cell subsets in the pathogenesis of ITP.Mechanism and treatment of patients with ITP to the high concentration of dexamethasoneare discussed. These results provide important information for understanding thepathogenesis and treatment of ITP.更多还原