TLR-9/MyD88信号通路在继发性噬血细胞性淋巴组织细胞增多症中发病机制研究

Hemophagocytic lymphohistiocytosis (HLH) is a clinical syndrome characterized by an uncontrolled proliferation of an activated monocyte-macrophage system that secretes high amounts of inflammatory cytokines and causes severe inflammation. Continual activating of TLR-9/MyD88 signal pathway can result in large amounts of cytokine release. However, whether this pathway is involved in the pathogenesis of sHLH is unclear. Our previous study demonstrated that peripheral CD14+ monocyte-macrophage TLR-9 mRNA in sHLH patients were significantly higher than those in healthy controls (p<0.05); The sTLR-4 in the peripheral blood were higher than those in healthy controls, too (p<0.05). In addition, we also found that peripheral blood CD4+CD25+CD127-Treg were higher than those in healthy controls and the level of Treg cells in post-treatment patients was significantly reduced compared to those pre-treatment (p<0.05). Based on these findings, we purpose the following hypothesis: (1): Whether TLR-9/MyD88 in monocyte-macrophage system activation is directly involved in the occurrence of sHLH? (2): Whether TLR-9 / MyD88 activation may affect the sHLH progression by regulating the level and function of Treg? To address these issues, we will use RAW264.7 monocyte macrophage cell lines, Treg of healthy controls, sHLH animal models, peripheral blood and bone marrow samples of sHLH patients, to further explore the pathogenesis and molecular mechanisms of TLR-9/MyD88 signaling pathway in sHLH. Additional methods such as Flow cytometry, RT-PCR, Western-Blot, and Co-immunoprecipitation will also be used in this study.

TLR-9/MyD88信号通路的持续激活可产生大量炎症因子造成机体各器官组织损伤，该信号通路是否参与继发性噬血细胞性淋巴组织细胞增多症（sHLH）的发病尚不清楚。我们发现sHLH患者外周血CD14+单核-巨噬细胞内TLR-9 mRNA、外周血sTLR-4、外周血的CD4+CD25+CD127-Treg均较正常对照组显著升高（p<0.05），TLR-9 mRNA和Treg在有效治疗后下降（P<0.05），提出假说：1. TLR-9/MyD88在单核-巨噬细胞系统内的激活直接参与sHLH的发生？2. TLR-9/MyD88可能通过调控Treg的数量和功能影响sHLH的疾病过程？我们用RAW264.7细胞系、正常供者Treg、sHLH动物模型及患者外周血和骨髓，采用FCM、RT-PCR、Western-Blot及免疫共沉淀等方法，探索持续TLR-9/MyD88信号通路活化在sHLH中的致病机制。

TLR-9/MyD88信号通路的持续激活可产生大量炎症因子造成机体各器官组织损伤,该信号 通路是否参与 sHLH的发病尚不清楚。通过研究我们证明了：1) CD14+单核细胞TLR-4分子的阳性表达率在sHLH患者中明显升高,尤其在IHLH组中高于AHLH、LHLH组,这对于鉴别诊断IHLH有重要作用,动态检测该指标有助于判断病情变化。2)初诊sHLH患者血清TRAF-6水平显著高于正常对照,获得临床缓解后血清TRAF-6水平较前显著下降。提示血清TRAF-6在sHLH患者的诊断及疗效评估等方面具有重要意义,TRAF6/NF-kB可能为TLR分子在sHLH发病机制中可能涉及的信号通路。3)miR-146a和miR-30e表达水平在sHLH患者显著降低,并且可以有效的鉴别出sHLH患者和正常人。血清中miR-30e与miR-146a表达水平比值(miR-30e/miR-146a)能够提示sHLH患者预后,比值低的sHLH患者预后可能较好。血清miR-146a表达水平与CD8+ T细胞的比例呈正相关,miR-146a低表达组的CD8+ T细胞的比例显著低于血清miR-146 a高表达组,因此miR-146a可能参与sHLH 发病机制中的CD8+ T细胞的缺失。4)PET-CT在无病理依据的sHLH的病因学判断中具有重要意义，并可为治疗方案的选择提供重要依据。该相关研究的结果目前已发表在SCI杂志,影响因子为3.14，发表36个月内被引用5次。5)HLH病程中常常伴随毛细血管渗漏综合征（CLS）的发生。严重的血小板、纤维蛋白原、白蛋白及钙离子减低,而三酰甘油、sCD25 及乳酸脱氢酶升高,可能是sHLH发生CLS的不良因素。sHLH患者合并CLS时预后差。积极治疗原发病、合理补液及供氧是关键,可有效控制疾病进展。6)血清钙离子浓度及白蛋白水平在sHLH患者的诊断、指导治疗及疗效监测等方面具有重要意义。7)探讨了sHLH患者胸腔积液的发生率、与其他实验室指标的相关性及其对生存率的影响。得出结论：胸腔积液对sHLH患者的诊断、病情评估和监测具有重要意义。