再生障碍性贫血患者骨髓自然杀伤T细胞体外活化特性研究

In vitro activation of bone marrow natural killer T cells of aplastic anemia patients

目的 研究再生障碍性贫血（再障）患者骨髓中自然杀伤T（NKT）细胞的数量以及体外活化后的功能状态.方法 将扩增活化骨髓单个核细胞（BMMNC）中NKT细胞的培养体系分为2组：①加入α-半乳糖神经酰胺（α-galactosylceramide,α-Galcer）＋rhIL-2;②加入α-Galcer＋rhIL-2＋rhGCSF.以流式细胞术（FACS）检测再障患者及正常对照组BMMNC中TCRVα24＋Vβ11＋细胞（NKT细胞）扩增前后的比率,同时测定NKT细胞活化后胞内IFN-γ、IL-4的表达.结果 再障患者组BMMNC中NKT细胞的比率为（0.19±0.09）%,NKT细胞在①、②培养体系中扩增活化7 d后,扩增倍数分别为79.91±40.56和67.45±29.42（P＜0.05）;活化后NKT细胞胞内IFN-γ阳性的比率分别为（62.31±14.67）%和（37.45±7.89）%（P＜0.01）;IL-4阳性的比率分别为（27.03±9.88）%和（55.11±12.13）%（P＜0.01）.正常对照组BMMNC中NKT细胞的比率为（0.25±0.12）%,较再障患者组显著升高（P＜0.05）,正常对照组NKT细胞在相应的①、②培养体系中的扩增倍数均高于再障患者组（P＜0.05）;活化后NKT细胞胞内IFN-γ的表达均低于再障患者组（P＜0.05）.结论 再障患者骨髓中NKT细胞较正常对照显著减少,扩增能力较正常对照降低,活化后IFN-γ的表达较正常对照升高.在rhG-CSF作用下,再障患者骨髓中NKT细胞经α-Galcer刺激后的扩增能力下降,表达IL-4的NKT2型细胞优势扩增,IL-4表达及分泌增加.

Objective To investigate the quantitative and qualitative changes of TCRVα24＋Vβ11＋natural killer T （NKT） cells from bone marrow （BM） of aplastic anemia （AA） after in vitro stimulation of α-galactosylceramide （α-Galcer）. Methods NKT cells in the bone marrow mononuclear cells （BMMNCs） from either AA patients or healthy controls were enumerated with flow cytometry. BMMNCs were cultured in RPM11640 medium supplemented with either α-Galcer and rhlL-2 or α-Galcer, rhIL-2 and rhG-CSF. The proliferative capacity of NKT cells was determined by NKT cell numbers before and after in vitro culture. Expression of intracellular IFNγ and IL-4 in activated NKT cells was analyzed with flow cytometry. Results In AA group, the percentage of NKT cells in BMMNCs was （0. 19±0.09）%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium resulted in significantly reduced expansion of NKT cells （67.45±29.42-fold vs 79.91±40.56 fold, P 〈0.05 ）. Meanwhile, addition of rhG-CSF reduced IFNγ positive NKT cells [（37.45±7.89）% vs （62.31±14.67）%, P〈0.01] and increased IL-4 positive NKT cells [（55.11±12.13 ）% vs （27.03±9.88 ）%, P 〈 0. 01 ]. In healthy control group, the percentage of NKT cells in B MMNCs was （0.25±0. 12）%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium also significantly reduced expansion of NKT cells（97.91±53.22 -fold vs 119.58±60.49 -fold, P 〈 0. 05 ）, reduced IFNγpositive NKT ceils [ （28.65±10.63）% vs （50.87±12.66）%, P〈0. 01 ], and increased IL-4 positive NKT cells[（66.53±14.96）% vs （31.11±10.07）%, P〈0.01]. Conclusion Compared to those from healthy controls, BMMNCs from AA patiants have a reduced fraction of NKT cells, which possesses a decreased potential to expand in vitro in response to ot-Galcer stimulation, and produce more IFNγ＋ NKTI cells, rhG-CSF, in combination with a-Galcer, confers polarization of NKT cells towards IL-4＋ NKT2 subpopulation.