铁过载对骨髓Coombs试验阳性全血细胞减少症患者骨髓造血损伤的机制研究

The mechanisms underlying bone marrow damage by iron overload in pancytopenic patients with positive BMMNC-Coombs test

目的探究铁过载对骨髓单个核细胞（BMMNC）Coombs试验阳性全血细胞减少症（又称免疫相关性全血细胞减少症，IRP）患者骨髓造血损伤的机制。方法采用流式细胞术检测21例铁过载｜RP患者、26例非铁过载IRP患者及10名正常对照者骨髓造血细胞活性氧自由基（ROS）、Bcl-2、Caspase-3水平及凋亡情况；并检测铁过载组BMMNC经N一乙酰半胱氨酸处理后上述指标的变化；同时检测IRP患者T细胞数量、比例及凋亡率。结果铁过载组BMMNC、粒系、红系和干细胞ROS水平及凋亡率均明显高于非铁过载组及正常对照组（P〈0．05）；铁过载组BMMNC、红系和干细胞Bcl-2水平均明显低于非铁过载组（P〈0．05）；铁过载组粒系、红系和干细胞Caspase．3水平均明显高于非铁过载组及正常对照组（P〈0．05）。铁过载组经N一乙酰半胱氨酸处理后，BMMNCROS、Caspase．3水平及凋亡率均较前明显下降，Bcl一2水平较前明显升高。铁过载组外周血CD4’细胞、CD4’／CD8’细胞比值分别为（40．86±8．74）％及1．44±0．36，均明显高于非铁过载组[（35．96±7．03）％及1．14±0．37]和正常对照组[（28．00±6．73）％及0．79-4-0．21]（P〈0．05），铁过载组CD8’细胞百分率明显低于非铁过载组和正常对照组（P〈0．05）。铁过载组骨髓CD8’细胞凋亡率、CD8‘细胞凋亡＊／CD4’细胞凋亡率比值分别为（27．35±10．76）％及2．52±0．81，均明显高于非铁过载组[（15．47±8．99）％及1．39±0．47]（P〈0．05）。在骨髓有核红细胞膜抗体或干细胞膜抗体阳性IRP患者中，铁过载组骨髓红系或干细胞凋亡率均高于非铁过载组（P〈0．05）。结论铁过载可能通过以下机制影响IRP患者骨髓造血功能：①过量铁沉积可诱导骨髓造血细胞ROS生成增加，影响Bcl一2、Caspase一3凋亡信号的表达，引起IRP患者骨髓造血细胞凋亡增加；②铁过载可通过影响T细胞数量及比例加重IRP患者免疫异常；③铁过载可增加IRP患者被覆自身抗体的骨髓有核红细胞和干细胞的破坏。

Objective To investigate the mechanisms underlying bone marrow damage by iron overload in pancytopenic patients with positive BMMNC-Coombs test （IRP）. Methods Twenty-one iron overloading, 26 non-iron overloading IRP patients and 10 normal controls were enrolled in this study. The expressions of ROS, Bcl-2, Caspase-3 and apoptosis of BMMNC were analyzed by flow eytometry （ FCM ）. Antioxidants were added to iron overloading IRP BMMNC, and then the changes of indices above were detected by FCM. The number and apoptosis of T lymphocytes of IRP patients were also detected. Results ROS and apoptosis of BMMNC, myelocytes, erythrocytes and stem cells of iron overloading IRP patients were significantly higher than that of non-iron overloading IRP ones and normal controls （ P 〈 0. 05 ）. The expressions of Bcl-2 on BMMNC, erythrocytes and stem cells of iron overloading IRP patients were significantly lower than those of non-iron overloading IRP ones （ P 〈 0.05 ）. The levels of Caspase-3 on myelocytes, erythrocytes and stem cells of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP ones and normal controls （ P 〈 0. 05 ）. After treatment with antioxidants, the expressions of ROS, Caspase-3 and apoptosis of iron overloading IRP BMMNC significantly decreased, but opposite for Bcl-2. The percentages of CD4 ＋ lymphocytes [ （40.86 ＋ 8.74）% ] and CIM＋/CD8 ＋ （1.44 ＋ 0.36） in PB of iron overloading IRP patients were significantly higher than that of non-iron overloading IRP ones [ （ 35.96 ＋ 7.03 ） % and 1.14 ＋ 0.37 ] and normal controls [ （ 28.00 -＋ 6.73 ） % and 0.79 ＋ 0.21 ], re- spectively （ P 〈 0.05 ） , as opposite for CD8 ~ lymphoeytes （ P 〈 0.05 ）. The apoptosis of CD8 ~ lymphoeytes [（27.35 ＋ 10.76）% ] and the ratio of CD8 ＋ apoptosis/CIM~ apoptosis （2.51 ＋ 0. 81 ） in BM of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP ones [ （ 15.47 -＋ 8.99 ） % ] and normal controls （ 1.39 ＋ O. 47 ） , respeetively （ P 〈 0.05 ）. The apoptosis of erythrocytes and stem cells eoated with auto-antibodies in BM of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP and normal controls. Conclusion Mechanisms underlying bone marrow damage by iron overload might be through the follows： @The inereased ROS induced by excessive iron depo- sition affected the expressions of Caspase-3 and Bcl-2, which caused more BMMNC apoptosis; @ The abnormal number and ratio of T lymphoeytes caused by iron overload aggravated the abnormality of immunity of IRP; Iron overload may increase the damage to erythrocytes and stem cells coated with auto-antibodies.