异常T细胞亚群参与免疫性血小板减少症的机制研究

发布时间：2018-07-02 09:27

本文选题：免疫性血小板减少症 + T细胞亚群　；参考：《苏州大学》2013年博士论文

【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。
[Abstract]:Immune thrombocytopenia (ITP) is a autoimmune disease characterized by immune destruction of platelets. It is divided into chronic ITP and acute ITP, and chronic ITP is frequently occurring between 20~50 years, the incidence of women is higher than that of men, and the majority of chronic ITP patients lack clinical manifestation or clear disease. Acute ITP is the most common hemorrhagic disease in children. The age of onset is more than 2~5 years old, and the course of disease is generally 4~6 weeks. Most of the sick children can be relieved by themselves. They seldom relapse after recovery. Most of them occur after virus infection or inoculation. Adults are rare and have no sex differences. Usually, there are more diseases in the peak period of the virus infection in winter and spring. According to clinical experience, most of the acute ITP can be naturally remission, and the chronic ITP disease is concealed and the cause is complex. More and more evidence shows that there is an imbalance of T cell subsets in ITP patients except for the disturbance of the humoral immune function and the production of their own anti platelet antibodies, such as the imbalance of Th1/Th2 and the abnormal expression of Th1, and the other studies have shown that Treg and The number of Th17 cells was abnormal.
The current clinical treatment of ITP is mainly through three ways to increase the number of platelets: (1) by inhibiting the clearance of platelets, such as splenectomy; (2) by inhibiting or correcting abnormal immune responses, such as steroid drugs or rituximab; (3) by increasing the formation of platelets, such as TPO-RAs., In some chronic ITP patients, the treatment effect is not significant.
It is reported that short range and large dose of dexamethasone has better therapeutic effects on chronic ITP patients, but the mechanism is unclear. This study tried to study the therapeutic effect of dexamethasone on ITP patients and elucidate the mechanism of its action. First, the flow formula of T cell subsets in peripheral blood of children with ITP, including Th1, Th2, Treg and Th17 cells was carried out. Analysis and ELISA determination of cytokines such as IL-2, IFN- gamma, IL-4, IL-10, TGF beta and IL-17. The changes of T-bet, GATA-3, Foxp3 and ROR T were analyzed in real time. Use the mechanism.
We performed a flow analysis of Th1, Th2, Treg and Th17 in 30 normal controls and 30 children with ITP. The results showed that there was no significant change in Th1 cells in normal controls and children with ITP, but the expression of Th2 cells in ITP decreased significantly. The number of Treg cells in ITP children decreased and the number of Th17 cells increased. The results showed that the cytokine (IL-2 and IFN- gamma) of Th1 increased significantly in the peripheral blood of children with ITP, while the content of the cytokines (IL-4 and IL-10) of Th2 decreased obviously. The expression of TGF beta and IL-17 was in accordance with the results of Treg and Th17 cells, which were decreased and increased respectively. There was no significant change in the expression of GATA-3 and Foxp3, but the expression of ROR and t increased significantly in ITP patients.
10 ITP patients were treated with short range and large dose of dexamethasone. The results showed that the number of platelets increased significantly in 70% of the patients and reached the normal level. We analyzed the T cell subsets and transcription factors. The results showed that the peripheral blood Th2 cells and Treg cells in the patients with dexamethasone were compared with those in the untreated group. In order to further reveal the mechanism of using dexamethasone to correct the T cell subpopulation imbalance in the treatment of ITP, we purify and separate normal T cells, and deimethasone in vitro. The results show that dexamethasone can inhibit the expression of Th17 cells. The expression of ROR gamma t contributes to the expression of GATA-3 and Foxp3. These results suggest that dexamethasone regulates the differentiation of T cell subsets by regulating the expression level of transcription factors to correct the abnormal state of T cell subsets in ITP patients, thus achieving the purpose of treating ITP.
To sum up, we first analyzed the expression of T cell subsets, cytokines and transcription factors in children's ITP, and provided more evidence for the involvement of abnormal T cell subsets in the pathogenesis of ITP, and the mechanism of the treatment of ITP patients with large dose of dexamethasone was preliminarily discussed in order to reveal the pathogenesis of ITP. And it provides important information for the treatment of ITP.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R725.5

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