骨髓NKT细胞活性与儿童再生障碍性贫血相关性研究

Bone marrow natural killer T cell activity in children with aplastic anemia

目的探讨骨髓中自然杀伤T细胞(nature killer T cell,NKT)在再生障碍性贫血免疫介导发病机制中的作用。方法选取14例确诊儿童再生障碍性贫血为再生障碍性贫血组,同年龄正常儿童10例为对照组。流式细胞术检测两组儿童骨髓中NKT含量;利用免疫磁珠法分离纯化NKT细胞后,分别在4种不同体系下进行扩增培养。A:α-Galcer+rhIL-2;B:α-Galcer+rhIL-2+rhG-CSF;C:OCH+rhIL-2;D:OCH+rhIL-2+rhG-CSF。测定NKT细胞在不同培养条件下的扩增倍数;利用酶联免疫斑点技术(Elispot)测定NKT细胞扩增活化后表达IFN-γ、IL-4的斑点形成细胞数(SFCs)。结果再生障碍性贫血组骨髓中NKT百分率(0.827%±0.022%)明显低于对照组(1.033%±0.073%,Z=-3.810,P=0.000)。在各培养体系中,再生障碍性贫血组和对照组骨髓NKT均可明显扩增。在含有α-Galcer培养体系A和B中,再生障碍性贫血组及对照组NKT细胞的扩增能力均高于OCH体系C和D,差异有统计学意义(P<0.01或P<0.05)。在培养体系中加入rhG-CSF后,再生障碍性贫血组骨髓NKT细胞的扩增能力下降(P<0.01),但表达IFN-γ的SFCs明显减少(P均<0.01);而表达IL-4的SFCs明显升高(P均<0.01)。结论再生障碍性贫血患儿骨髓NKT的数量和功能均明显低于正常儿童。但再生障碍性贫血骨髓在接受细胞配体(OCH或α-Galcer)+rhIL-2+rhG-CSF作用后,在获得NKT细胞一定程度扩增的同时,提高NKT的IL-4表达,降低NKT的IFN-γ表达,抑制T细胞向Th1分化,而促进T细胞向Th2分化,逆转再生障碍性贫血Th1/Th2异常失衡的免疫介导致病机制,可能成为获得性再生障碍性贫血的有效治疗途径。

Objective To investigate activity of bone marrow nature killer T cell （NKT） and its relation to pathogenesis of aplastic anemia（AA） in children. Methods Fourteen children with aplastic anemia were included in the study and 10 healthy children were selected as controls. The proportion of NKT cells in bone marrow was measured by flow cytometry. Immune magnetic bead separation was used to isolate and purify NKT cells. The purified NKT cells were cultured in four different culture systems. A： α-Galcer and rhlL-2 ; B ： α-Galcer, rhIL-2 and rhG-CSF ;C ： OCH and rhlL-2 ; D ： OCH, rhlL-2 and rhG-CSF. The amplification of NKT cells after cultured in different systems was calculated. Elispot method was used to analyze the sporing form cells （SFCs） expressing IFN-γ or IL-4. Results The percentage of NKT cells in bone marrow of AA group （ 0. 872%± 0. 022% ） was significantly lower than control group （ 1. 033 % ± 0. 073 %, Z = - 3.810, P = 0. 000 ）. In each culture system, the bone marrow NKT cells in both groups were significantly amplified. In system A and B containing α-Galeer, the proliferation ability of NKT ceils from both AA group and control group were higher than that in C and D （ OCH system） （ P 〈 0.01 or P 〈 0.05 ）. rhG-CSF decreased the expansion of NKT cells but resulted in decreased SFCs of IFN-γ and elevated SFCs of IL-4 in AA group（P 〈0.01 ）. Conclusion The quantity and function of NKT cells from children with aplastic anemia are lower than those from healthy children. But NKT cells can be amplified and its function can be improved by interaction with cell ligand OCH or α-Galcer, rhIL-2 and rhG-CSF, which might be a potential therapeutic approach for AA.