重型再生障碍性贫血细胞毒性T细胞免疫攻击靶点的体外实验

Target cells of cytotoxic T ceils in severe aplastic anemia in vitro

目的对重型再生障碍性贫血（SAA）患者细胞毒性T细胞所攻击的靶细胞以及各系、各阶段骨髓造血细胞的凋亡配体表达进行研究，为明确SAA免疫攻击的靶点提供依据。方法选取2011年3月至2012年3月天津医科大学总医院血液科收治的SAA患者15例，以15份正常骨髓标本作为对照，应用流式细胞术（FCM）检测骨髓CD34＋、CD14＋、CD33＋、GlycoA＋细胞凋亡相关因子（Fas）蛋白的表达量。免疫磁珠分选SAA患者骨髓中CD8＋T细胞和对照骨髓中去除CD3＋细胞的骨髓单个核细胞（靶细胞），并进行共培养，FCM分析CD34＋、CD14＋、CD33＋、GlycoA＋细胞群凋亡率。结果SAA患者骨髓CD34＋细胞Fas蛋白的表达量（46．59％±27．60％）明显高于对照骨髓（8．89％±7．28％，P〈0．01）；SAA患者骨髓CD14＋、CD331＋、GlycoA＋细胞Fas蛋白的表达量（29．29％±9．23％、46．88％±14．30％、15．15％±9．26％）明显低于对照骨髓（51．25％±38．36％、72．06％±39．88％、50．38％±39．88％，均P〈0．05）。体外实验中，实验组（SAA患者骨髓CD8＋T细胞与对照骨髓CD3-细胞混合培养组）的CD34＋、CD33＋、CD14＋细胞凋亡率（55．43％±20．50％、38．13％±20．10％、61．87％±21．65％）均明显高于对照组（对照骨髓CD8＋T细胞与对照骨髓CD3-细胞混合培养组）（35．02％±13．95％、23．44％±10．33％、37．04％±22．41％，均P〈0．05）。结论SAA细胞毒性T细胞对正常的骨髓造血干／祖、粒系、单核系细胞均具有杀伤作用。Fas／Fas配体系统介导的细胞凋亡在SAA免疫发病中发挥了重要作用。同时CD34’细胞Fas表达明显增加，可能是SAA免疫损伤的主要靶细胞。

Objective To clarify the specific target of severe aplastie anemia （SAA） immune attack via identifying the target cells of cytotoxic T cell attacks and the expression of apoptosis ligand on each department and each stage of bone marrow hematopoietic ceils. Methods A total of 15 SAA patients and 15 normal controls were recruited in the Department of Hematology, Tianjin Medical University General Hospital between March 2011 and March 2012. Factor associated suicide （Fas） protein expression of CD34 ＋ , CD14 ＋ , CD33 ＋ , and GlycoA ＋ cells in bone marrow was detected by flow cytometry. The CD8 ＋ T ceils of SAA patients and CD3- bone marrow inononuelear cells （BMMNC） of controls were sorted by immunomagnetie separation and co-cultured for 72 hours. The apoptosis rate of CD34 ＋ , CD14 ＋ , CD33 ＋ , and GlycoA ＋ cells were measured with flow cytometry. Results The expression of Fas protein in CD34 ＋ cells in SAA patients （46.59% ± 27.60% ） was significantly higher than that in control group （8.89% ± 7.28%, P 〈0. 01 ）. The expressions of Fas protein in CD14＋, CD33 ＋ and GlycoA＋ ceils in SAA group （29.29% ± 9. 23% , 46. 88% ± 14. 30% , 15. 15% ± 9. 26% ） were lower than those in control group （51.25% ±38.36%, 72. 06% ±39.88%, 50. 38% ±39.88%, all P〈0.05）. The apoptosis rates of CD34 ＋ , CD33 ＋ and CD14＋ cells in the experimental group （ CD8 ＋ T cells of SAA patients co-euhured with CD3- BMMNC of controls： 55.43% ± 20. 50%, 38.13% ± 20. 10%, 61.87% ± 21.65%） were significantly higher than those of the control group （ CD8＋ T cells of controls co-cultured with CD3 - BMMNC of controls： 35.02% ± 13.95%, 23.44% ± 10. 33%, 37. 04% ± 22. 41%, all P 〈 0.05 ）. Conclusions Cytotoxic T ceils in SAA patients may have a killing effect on hematopoietie stem/progenitor cells, and granulocytic and macrophagocytic cells from norma/ bone marrow. Moreover, Fas/Fas ligand-mediated apoptosis may play an important role in the immune pathogenesis of SAA. CD34＋ cells show markedly increased Fas protein expression, which may be the main target ceils in the process of immune injury in SAA patients.