干扰素γ介导噬血细胞综合征血细胞减少的机制研究

Hemophagocytic syndrome (HPS) is a group of immune dysfunction caused by excessive inflammation, and reduction of peripheral blood cells is the most important characteristic of the disease. Our research group found that difficult correction of progressive pancytopenia is closely related with poor prognosis. Thus, effectively improving the state of the level of peripheral blood cells, shortening the duration of the reduction of blood cells is a key part of treatment. The level of serum interferon-γ (IFN-γ) is correlate with the reduction of blood cells. The pre-experimental results prompted it may affect the differentiation of CD34+ cells, which decreased the production of blood cells and mediated macrophage activation to generate hemophagocytic phenomenon and then accelerate consumption of blood cells. The subject is to be adopted by cell growth in vitro culture and in vivo studies which using flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assay and Western blot method to confirm this hypothesis. Initially CD34+ cells are cultured in vitro and observe the impact of IFN-γ on cell differentiation; then observe of the effect of IFN-γ on the number of blood cells in gene-deficient mice model and the identify the function of macrophage and the signal transduction pathways of IFN-γ receptor. The above study is designed to clarify the pathophysiology of IFN-γ-mediated cytopenia of HPS and provide a theoretical basis for exploring the new clinical treatment.

噬血细胞综合征(HPS)是一组因免疫功能异常导致的过度炎症反应，外周血细胞减少是该病最主要的特点。本课题组研究发现进行性难以纠正的全血细胞减少与不良预后密切相关。有效改善外周血细胞水平状态，缩短血细胞减少持续时间是治疗的关键环节。血清干扰素γ(IFN-γ)水平与血细胞减少存在相关性，预实验结果提示其可能影响CD34+细胞的分化，使血细胞生成减少，并介导巨噬细胞活化产生噬血现象加速血细胞消耗。为证实这一假说，本课题拟通过体外细胞培养及动物体内试验，采用流式细胞术、免疫组化、酶联免疫吸附试验及免疫印迹方法，首先将CD34+细胞体外培养，观察IFN-γ对其分化的影响；其次在基因缺陷小鼠模型中观察IFN-γ对血细胞数量的影响，并鉴定巨噬细胞功能以及IFN-γ受体信号传导通路。上述研究旨在阐明IFN-γ介导HPS血细胞减少的病理生理机制，为指导临床用药，探索治疗新方法提供理论依据。

本课题严格按照课题任务书计划开展相关研究，入组确诊HPS的初诊患者和健康志愿者共20例。分离纯化正常人和HPS患者的骨髓CD34+细胞，分别在含有G-CSF+IFN-γ、EPO+IFN-γ以及TPO+IFN-γ的体系中培养，观察CD34+细胞的定向分化情况，发现HPS患者骨髓与正常人骨髓在半固体培养基培养14天后分化情况未见差异。在上述各种培养体系中加入不同浓度的IFN-γ，观察不同浓度梯度IFN-γ对CD34+细胞定向分化的影响，发现当IFN-γ浓度提高到1ng/ml，骨髓细胞的分化开始出现影响，当IFN-γ浓度提高到10ng/ml时出现较明显影响。筛选出能够使CD34+细胞分化发生改变的最低IFN-γ浓度组，提高原体系G-CSF、EPO及TPO的浓度，观察CD34+细胞的定向分化情况，发现在IFN-γ浓度为10ng/ml时，提高G-CSF、GM-CSF及EPO的浓度，骨髓生长情况均较原体系好转，且以红系变化最为显著。本研究结果证实IFN-γ引起持续的血细胞减少与IFN-γ干扰CD34+细胞的定向分化有关，提高造血生长因子水平能够部分克服IFN-γ对CD34+细胞分化的影响。为进一步阐述论证HPS过程中血细胞生成减少的机制提供依据。. 在本课题研究基础上发表SCI论文2篇(已接受)，核心期刊论文3篇；全国性学术会议特邀报告3次，中国实验血液学年会分组报告1次；申请发明专利1项(已进入复审阶段)；由本课题组承办全国性学术会议1次；培养毕业硕士研究生2名，青年学术骨干2名。