中文摘要：

目的探讨NKT细胞数量及其功能在重型再生障碍性贫血患儿外周血及骨髓中的变化。方法采用流式细胞术检测10例重型再生障碍性贫血初治患儿及10例骨科对照儿童外周血及骨髓CD3＋CD1d四聚体＋NKT细胞水平。利用免疫磁珠法分离纯化iNKT细胞后,在NKT细胞的配体OCH＋重组人白介素-2（rh IL-2）＋重组粒系集落刺激因子（rhGCSF）培养体系下进行扩增培养。测定在不同浓度OCH培养条件下,iNKT细胞的扩增倍数,并利用酶联免疫斑点技术测定iNKT细胞扩增活化后表达肿瘤坏死因子（IFN-γ）、白细胞介素（IL-4）的斑点形成细胞数。结果再生障碍性贫血患儿外周血中CD3＋CD1d四聚体＋NKT百分率为（0.72±0.03）%,明显低于对照组的（0.92±0.02）%,骨髓中CD3＋CD1d四聚体＋NKT百分率为（0.82±0.02）%,明显低于对照组的（1.05±0.05）%,差异有统计学意义（P均=0.000）。再生障碍性贫血患儿骨髓iNKT细胞体外扩增能力显著低于对照组;且中高浓度OCH条件下,iNKT细胞扩增倍数增高,生成IFN-γ降低,而生成IL-4增高,与低浓度条件下相比,差异有统计学意义（P均〈0.01）。结论重型再生障碍性贫血患儿存在外周血及骨髓NKT细胞水平降低及功能异常;OCH能够促进iNKT细胞扩增,并可改善IL-4/IFN-γ的失衡,可能具有潜在的治疗价值。

英文摘要：

Objective To analyze the percentage and functional changes of natural killer T（NKT） cells in peripheral blood and bone marrow of severe aplastic anemia（SAA） children before immunosuppressive therapy（IST） comparing to that of healthy children. Methods Ten children with severe aplastic anemia were included in the study and ten healthy children at the same age were selected as the control group. By flow cytometry, the percentage of CD3＋CD1d tetramer＋ NKT cell in peripheral blood and bone marrow were detected from March 2014 to December 2014 in our hospital. Immune magnetic bead separation was used to isolate and purify i NKT cells.The purified i NKT cells were cultured in the OCH（50 ng/ml, 100 ng/ml or 200 ng/ml） ＋ rh IL-2 ＋ rh G-CSF culture systems. The amplification of i NKT cells after cultured in different systems were calculated. Elispot method was used to analyze the spotting form cells（SFCs） of IFN-γ or IL-4 expressed by activated i NKT cells. ResultsThe percentage of CD3＋CD1d tetramer＋ NKT cells in peripheral blood of SAA group（0.72±0.03）% was significantly lower than that of the control group（0.92±0.02）%（P=0.000）. The percentage of CD3＋CD1d tetramer＋ NKT cells in bone marrow of SAA group（0.82±0.02）% was significantly lower than that of the control group（1.05±0.05）%（P=0.000）. In vitro i NKT cell amplification ability of bone marrow in SAA group was significantly lower than the control group, and in medium concentration（50±6） and high concentration OCH group（52±6）, the amplification ability was higher than that in low concentration OCH group（30±5）（P〈0.05）. The secretion of IFN-γ in the i NKT cells of SAA bone marrow was significantly lower in medium concentration（33±3） and high concentration（35±3） OCH group than that of the low concentration（50±3） OCH group（P〈0.01）. The secretion of IL-4 in the i NKT cells of SAA bone marrow was significantly higher in medium concentration（50±3） and high concentrat