抗CD44单克隆抗体IM7在体外诱导慢性髓系白血病干／祖细胞的凋亡

Apoptosis of Chronic Myeloid Leukemia Stem/Progenitor Cells Induced by Anti-CD44 Monoclonal Antibody IM7 In Vitro

本研究探讨抗CD44单克隆抗体IM7在体外诱导慢性髓系白血病干/祖细胞(leukemic stem/progenitor cells,LSPC)凋亡情况及其可能的作用机制。取20例初诊的慢性髓系白血病(chronic myeloid leukemia,CML)病人骨髓液5-10ml,用免疫磁株分离法分离出CD34+、CD38-、CD123+LSPC。利用Annexin-V试剂盒检测IM7诱导CML-LSPC凋亡情况;荧光实时定量PCR及RT-PCR检测CML-LSPC中原癌基因c-myc、NF-κB表达水平;Western-blot检测IM7孵育后CML-LSPC中BCL-2蛋白表达变化。结果表明:经IM7孵育后CML-LSPC的早期凋亡率由对照组(5.42±1.84)(升高至(12.58±2.84)((p<0.05)。IM7孵育后CML-LSPC中原癌基因c-myc、NF-κB mRNA表达水平明显降低,IM7能有效抑制CML-LSPC中BCL-2蛋白表达下调,CML-LSPC中NF-κB的活性受到抑制。结论:抗CD44单克隆抗体IM7能有效诱导CML-LSPC凋亡,其可能机制为NF-κB的活性降低,作为下游靶基因c-myc及bcl-2表达下调,从而诱导LSPC凋亡。

The aim of this study was to investigate the apoptosis-inducing effect of anti-CD44 monoclonal antibody IM7 on chronic myeloid leukemia （CML） stem/progenitor cells in vitro and to explore its possible mechanism. Leukemic stem/progenitor cells （ LSPCs ） expressing CD34 ^＋ , CD38 - and CD123 ^＋ were isolated from bone marrow （BM） cells of 20 patients with newly-diagnosed chronic myeloid leukemia by using EasySepTM magnetic beads. The percentage of apoptotic CML-LSPCs was assayed by Annexin-V/PI staining; the expression changes of c-myc and NF- KB mRNA were detected by real-time quantitative PCR（RQ-PCR） and RT-PCR; the NF-KB activity was detected by NF-KB Activation Nuclear Translocation Assay Kit; the BCL-2 protein expression was determined in the Western blot method. The results showed that the IM7 effectively induced apoptosis of CML-LSPCs; the mean percentage of early apoptotic cells significantly increased, as compared with the untreated control CML-LSPCs cells 12.58 ± 2.84% vs 5.42 ±1.84% （p 〈 0.05 ）. The c-myc , NF-KB mRNA expressions were down -regulated as compared with the control group （0.65 ±0. 10 vs 1. （90, 0.42 ±0.21 vs 1.00, respectively） （p 〈0.01 ） by RQ-PCR and （0.49 ±0.09 vs 0. 60 ± 0.12, 0.47 ± 0.11 vs 0. 67 ± 0.08, respectively ） （p 〈 0.01 ） by RT-PCR. The BCL-2 protein level in CML-LSPCs treated with 1M7 also decreased as compared with the control group （p 〈0.01 ）. In addition, the depression of NF-KB activity was observed through fluorescence microscope. It is concluded that the anti-CD44 monoclonal antibody IM7 effectively induces apoptosis of CML-LSPCs through down-regulating c-myc and bcl-2 mRNA expression, and decreasing NF-KB activity in CML-LSPCs.