Bcl-2小分子抑制剂ABT-737逆转CD34+急性髓细胞白血病耐药及其机制的体内外研究

CD34 positive acute myeloid leukemia (CD34+AML) cells are resistant to current chemotherapies. CD34+AML cells are 10-15-fold more resistant to chemotherapies than CD34-AML cells. CD34+AML are responsible for relapse of leukemia. However, the mechanism underlying the resistance of CD34+AML to chemotherapies remains unclear. Our previous study demonstrates that CD34+AML cells have higher level of Bcl-2 protein expression than the CD34- subpopulation. These findings suggest that Bcl-2 plays a critical role in CD34+AML cell survival. In this study, we examined following contents: (1) CD34+AML cells and CD34-AML cells were sorted from both AML cell lines and AML patients by using MicroBead kit. To clarify if Bcl-2 overexpression plays an important role in drug-resistance, Bcl-2 expression was suppressed by siRNA in CD34+AML and the effects on chemotherapies sensitivity were examined. On the contrary, Bcl-2 expression was up-regulated by transfection in CD34-AML and the same effects were compared. (2) We examined the cytotoxic efficiency of small-molecule Bcl-2 inhibitor ABT-737 on CD34+AML cells both in vitro and in the NOD/SCID mice. These results are used to provide the therapy targets and the evidence for the ability of ABT-737 to overcome resistance to chemotherapies by down-regulation of Bcl-2 in CD34+AML cells.

CD34阳性表型急性髓细胞白血病(CD34+AML)对化疗耐药，其耐药性是CD34-AML的10-15倍，是AML复发难治的根本原因，但耐药机制不明。我们前期结果表明CD34+AML的Bcl-2表达显著高于CD34-AML，因此设想是否Bcl-2过表达导致CD34+AML耐药？本项目研究内容包括：(1)以AML细胞系和AML患者原代细胞为研究对象，采用免疫磁珠分选CD34+AML和CD34-AML细胞，RNA干扰沉默CD34+AML高表达的Bcl-2，基因转染上调CD34-AML低表达的Bcl-2，检测Bcl-2表达变化对化疗敏感性的影响，正反论证Bcl-2在耐药中的作用。(2)在细胞和荷瘤NOD/SCID小鼠中探讨Bcl-2小分子抑制剂ABT-737靶向治疗CD34+AML的作用。研究成果有望为治疗CD34+AML提供靶点，明确ABT-737逆转耐药、靶向治疗CD34+AML可行性。

研究背景：CD34阳性表型急性髓细胞白血病(CD34+AML)对化疗耐药，其耐药性是CD34-AML的10-15倍，是AML复发难治的根本原因，但耐药机制不明。我们前期结果表明CD34+AML的Bcl-2表达显著高于CD34-AML，因此设想是否Bcl-2过表达导致CD34+AML耐药？本项目研究内容：(1)采用免疫磁珠分选CD34+AML和CD34-AML细胞，RNA干扰沉默CD34+AML高表达的Bcl-2，基因转染上调CD34-AML低表达的Bcl-2，检测Bcl-2表达变化对化疗敏感性的影响，正反论证Bcl-2在耐药中的作用。(2)在细胞和荷瘤NOD/SCID小鼠中探讨Bcl-2小分子抑制剂ABT-737靶向治疗CD34+AML的作用。研究成果有望为治疗CD34+AML提供靶点，明确ABT-737逆转耐药、靶向治疗CD34+AML可行性。重要结果：(1)在细胞系以及原代细胞中均证实，CD34+AML的Bcl-2表达显著高于CD34-AML，通过Bcl-2 siRNA下调CD34+AML的Bcl-2表达后显著增加细胞对柔红霉素化疗敏感性，反之上调 Bcl-2基因可以降低CD34-AML细胞对柔红霉素敏感性。正反论证了Bcl-2在CD34+AML耐药中的作用；(2) ABT-737可抑制CD34+AML增殖、诱导其凋亡。ABT-737可增加KG1a和Kasumi-1细胞对柔红霉素、阿糖胞苷敏感性，协同发挥抗白血病作用。值得注意的是，ABT-737单药及联合柔红霉素可抑制原代CD34+AML细胞增殖、诱导其凋亡，发挥协同抗白血病作用，而对CD34+正常造血祖细胞活性无明显影响。(3) ABT-737对NOD/SCI小鼠白血病模型均具有显著肿瘤抑制作用，延长小鼠生存时间。科学意义：本课题深入探讨了Bcl-2在CD34+AML亚群的耐药机制，在细胞系、原代细胞及荷瘤小鼠中阐明了其小分子抑制剂ABT-737靶向抗白血病效应，为难治复发CD34+AML提供新的治疗方向和思路。