非经典性NF—κB活性在慢性B淋巴细胞白血病中的作用

Function of alternative NF-κB activity in B-cell chronic lymphocytic leukemia cells

目的探讨非经典性NF—κB信号通路在慢性B淋巴细胞白血病（B—CLL）中的作用。方法采用实时荧光定量RT-PCR法检测56例B—CLL初诊患者骨髓CD5＋CD19＋细胞（CLL—B细胞）NF-κB家族成员mRNA表达水平；采用TransAM^TM ELISA法定量分析CLL—B细胞核中NF-κB蛋白质复合物的活性成分；采用流式细胞术分析CLL—B细胞体外单独或与骨髓基质细胞（hBMSC）共培养后细胞死亡率；采用Westemblot法检测共培养对骨髓CLL-B细胞中NF-κB家族成员RelA和RelB蛋白水平表达的影响。结果CLL—B细胞的NF-κB家族成员RelA、p50、RelB和p52mRNA水平均高于正常B细胞，差异有统计学意义（P值均〈0．01）。所有标本中CLL—B细胞都存在RelA活化，而RelB只在部分标本中活性增强。CLL．B细胞核中平均RelA活性比正常B细胞显著增强，而平均RelB活性与正常B细胞相似。体外单独培养24、48和72h时，RelA＋／RelB组CLL．B细胞死亡率分别为（35．544-4．43）％、（50．92土8．44）％和（49．24士8．16）％；RelA＋／RelB＋组CLL—B细胞死亡率分别为（20．65±2．37）％、（18．17±1.36）％和（26．55±4．08）％。与hBMSC共培养，RelA＋／RelB一组CLL．B细胞死亡率较单独培养时下降，但相对于RelA＋／RelB＋组CLL—B细胞死亡率仍较高；共培养48h后，RelA＋／RelB-组CLL—B细胞中RelA和RelB显著上调，RelA＋／RelB＋组中CLL—B细胞质中RelA表达上调，RelB在胞质中显著下调并在胞核中轻度上调。结论骨髓CLL—B细胞中存在非经典性NF-κB的活化，RelB活化程度在不同的CLL—B细胞中差异显著；RelB和RelA活化共表达赋予骨髓CLL—B细胞存活优势，CLL—B细胞与hBMSC共培养，通过诱导CLL—B细胞中RelA和RelB的表达发挥对其体外存活的保护作用。

Objective To investigate the function of alternative NF-κB activity in B-cell chronic lymphocytic leukemia cells （B-CLL）. Methods The mRNA expression of individual NF-κB subunits in CD5＋CD19＋ cells （CLL B-cells） from bone marrow （BM） of 56 patients with B-CLL was analyzed by quantitative RT-PCR. An ELISA-based NF-κB family transcription factor activity assay was performed to quantify the κB DNA-binding activity in nuclear extracts from CLL B-cells. Cell death of CLL-B cells was determined by PI staining, RelA and RelB expression at protein level of CLL B-cells by Western blot analyses. Results The expression levels of RelA, pS0, RelB and p52 mRNA in CLL B-cells were all higher than that of normal B cells with statistical significance （P〈0.05）. RelA was activated in almost all the patients detected while RelB activity was induced in part of samples. The average RelA activity in CLL B-cells was increased compared to that in normal B cells while the average RelB activity was similar to that of normal B cells. When cultured in vitro for 24, 48 and 72 hours, the frequencies of cell death of CLL B-cells from RelA＋/RelB- group were （35.54±4.43）%, （50.92±8.44）%, and （49.24±8.16）%, respectively; that of the RelA＋/RelB＋ group were （20.65±2.37）%, （18.17± 1.36）%, and （26.55±4.08）%, respectively. When the cells from RelA＋/RelB-group were co-cultured with bone marrow stromal cells （hBMSCs）, the frequencies of cell death of CLL B-cells were decreased compared to that of the cells cultured alone, while the frequencies of cell death of RelA＋/RelB CLL B-cells were higher than that of CLL B-cells from RelA＋/ RelB＋ group when co-cultured with hBMSCs. RelA and RelB expression in CLL-B cells from the RelA＋/ RelB group was induced after co-cultured with hBMSCs for 48h. RelB was reduced in the cytoplasm and increased in the nucleus in CLL-B cells from the RelA＋/RelB＋ group. Conclusion The alternative NF-~B was indeed activated and presented heterogenous in CLL B-cells from BM. Activation of RelB combined with RelA activity could provide the survival advantage to CLL B-cells from BM. Co-culture with hBMSCs could protect CLL-B cells through the induction of RelA and Rel B expressions.