iNKT细胞通过免疫调控改善再生障碍性贫血骨髓衰竭的机制研究

The development of acquired aplastic anemia（AA） is primarily ascribed to activation and proliferation of T cells, and then T cells transform into Th1 cells, producing explosive inflammatory cytokines, such as IFN-γ and TNF-α. The autoimmune response may directly destroy hematopoietic stem and progenitor cells in bone marrow by activated Th1 cells. Constitutive expression of T-bet, a transcriptional regulator, is critical to induce Th1 polarization, which is commonly found in a majority of acquired AA patients. To suppress Th1 polarization may be open an avenue of effective treatments for human acquired aplastic anemia. Invariant natural killer T (iNKT) cells are characterized as unique sub-typed T lymphocytes. Recently, it was reported that an analog of α-galactosylceramide (α-GC), OCH, which has a truncated sphingosine chain, stimulates NKT cells to produce IL-4. Therefore, OCH has the potential to elicit protective immunity against Th1-mediated autoimmune disease..Our previous studies demonstrated that OCH ameliorated immune-mediated bone marrow failure syndromes（BMFS）in CByB6F1 mice via activation of NKT cells, and shifting the balance from Th1 to Th2. The expression of T-bet protein in spleen was lower in OCH treated BMFS mice than in BMFS controls. We also found that OCH can adjust the secretion of IL-4/IFN-γ in aplastic anemia children bone marrow iNKT cells in vitro. We speculated that regulatory iNKT cells may be a new target in the treatment of aplastic anemia..In the present study, we want to examine the effects of iNKT cells on immune-mediated acquired aplastic anemia children. This project aims to continue to explore: a.The change of number and function of aplastic anemia in children for different stages of iNKT cells, naive CD4+ T, Th1, Th2. The gene expression of iNKT cells by microarrays. The number, cell cycle and apoptosis of the HSC/HPC cells. b. Examine the Th1/Th2 and HSC/HPC cells by Flow cytometry for the number, cell cycle and apoptosis in immune-mediated bone marrow failure mouse model with iNKT cells without stimulation, with stimulation of OCH or α-GC. Investigate the relationship between the numbers and function of iNKT cells and the pathogenesis and prognosis of children aplastic anemia. Explore a new method of treatment for acquired aplastic anemia by finding the immune regulation mechanism of iNKT cells in aplastic anemia.

目前认为再生障碍性贫血是Th1细胞异常活化致造血干细胞损伤的一种自身免疫性疾病。稳定自然杀伤T细胞（iNKT）在调节Th1和Th2免疫平衡中起关键作用。课题组前期研究发现OCH（人工合成NKT配体）可通过活化NKT，抑制Th1异常活化，逆转免疫诱导的小鼠骨髓衰竭，下调脾脏T-bet表达，我们临床结果还发现OCH体外可调节再障患儿骨髓iNKT细胞IL-4/IFN-γ分泌，推测调节iNKT细胞可能是治疗再障新靶点。本项目拟研究：①再障儿童疾病不同阶段iNKT，naive CD4+ T，Th1，Th2细胞的含量；骨髓iNKT细胞的基因表达；造血干细胞的数目、细胞周期与凋亡的变化。②再障小鼠输入活化的iNKT细胞，观察其Th1/Th2变化，造血干细胞比例，细胞周期及凋亡的变化。解析iNKT细胞在骨髓细胞增生中的免疫调节机制，阐明iNKT细胞比例和功能与再障发病及预后的关系，探索治疗再障的新方法。

目前认为获得性再生障碍性贫血是Th1细胞异常活化致造血干细胞损伤的一种自身免疫性疾病。本研究通过免疫诱导骨髓衰竭小鼠模型及再障患儿的相关研究，进一步探讨再障的免疫致病机制。1.成功制备免疫诱导骨髓衰竭小鼠模型,并探索了一些免疫调节干预对骨髓造血的影响。2.分析比较不同剂量的静脉丙种球蛋白辅助治疗儿童获得性重型再障的疗效。研究显示免疫抑制治疗的获得性重型障患儿,高剂量 IVIG 辅助治疗可使早期反应率增加，但并未增加其远期有效率、治愈率及 5 年生存率；可减少严重感染率，但未能减少总感染率及感染相关死亡率。因此提出丙球在临床中需严格掌握适应症，避免不必要的经济浪费。3.分析再障患儿治疗前IL-6水平对免疫抑制治疗疗效的影响。研究显示治疗前高水平的IL-6可能预示应用免疫抑制治疗疗效相对好，利于患儿今后临床分层治疗。4.采集再障患儿与健康人的外周血进行转录组测序。利用生物信息学手段获得患儿与健康人之间基因差异表达，从mRNA 与 IncRNA 层面探索再障的致病机制。再障患儿相对健康儿童上下调基因KEGG通路富集分析，显示在上调基因显著富集通路中7个通路可能与再障相关；在下调基因显著富集通路中3个通路可能与再障相关。5.通过对再障骨髓间充质干细胞单细胞测序分析研究，显示IFN-γ 和TNF-α 短期刺激处理，可能导致骨髓间充质干细胞分析和表达一系列趋化因子、细胞因子和表面分子，起到免疫抑制的作用。6.总结分析临床患儿骨髓中Th1/Th2调控转录因子的表达及与临床相关性的研究。检测调节Th1/Th2细胞分化的T-bet，GATA3蛋白在不同程度再障患儿骨髓中的表达，同时比较外周血细胞因子水平，骨髓衰竭重的再障患儿其骨髓T-bet蛋白表达明显增高并细胞因子差异，调节Th1的T-bet蛋白在再障的免疫病理机制中可能起着一定作用。

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