CAJ | 학술논문

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.
목적 연구재생장애성빈혈(재장)환자골수중자연살상T(NKT)세포적수량이급체외활화후적공능상태.방법 장확증활화골수단개핵세포(BMMNC)중NKT세포적배양체계분위2조:①가입α-반유당신경선알(α-galactosylceramide,α-Galcer)+rhIL-2;②가입α-Galcer+rhIL-2+rhGCSF.이류식세포술(FACS)검측재장환자급정상대조조BMMNC중TCRVα24+Vβ11+세포(NKT세포)확증전후적비솔,동시측정NKT세포활화후포내IFN-γ、IL-4적표체.결과 재장환자조BMMNC중NKT세포적비솔위(0.19±0.09)%,NKT세포재①、②배양체계중확증활화7 d후,확증배수분별위79.91±40.56화67.45±29.42(P＜0.05);활화후NKT세포포내IFN-γ양성적비솔분별위(62.31±14.67)%화(37.45±7.89)%(P＜0.01);IL-4양성적비솔분별위(27.03±9.88)%화(55.11±12.13)%(P＜0.01).정상대조조BMMNC중NKT세포적비솔위(0.25±0.12)%,교재장환자조현저승고(P＜0.05),정상대조조NKT세포재상응적①、②배양체계중적확증배수균고우재장환자조(P＜0.05);활화후NKT세포포내IFN-γ적표체균저우재장환자조(P＜0.05).결론 재장환자골수중NKT세포교정상대조현저감소,확증능력교정상대조강저,활화후IFN-γ적표체교정상대조승고.재rhG-CSF작용하,재장환자골수중NKT세포경α-Galcer자격후적확증능력하강,표체IL-4적NKT2형세포우세확증,IL-4표체급분비증가.
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.