我们已证实Disulfiram螯合Cu（DS/Cu）可通过上调ROS、抑制NF-κB靶向杀伤AML干细胞。HIF-1α可通过SDF-1/CXCR4-VLA4影响AML干细胞与基质细胞的黏附作用，并受NF-κB通路调控。我们预实验发现DS/Cu可下调骨髓基质细胞与AML干细胞HIF-1α表达且对骨髓基质细胞无明显诱导凋亡作用。我们提出调控HIF1a通路、干扰AML干细胞微环境可能是DS/Cu靶向杀伤AML干细胞的一个新机制。拟利用已建立的AML干细胞-骨髓基质细胞共培养及AML干细胞NOD/SCID小鼠模型，通过免疫印迹、免疫组化等方法检测DS/Cu作用前后HIF-1α及相关细胞因子和黏附分子表达的变化，获得DS/Cu对AML干细胞微环境影响的直接证据；并深入探讨ROS及NF-κB通路对HIF-1α的调节作用，阐明DS/Cu靶向杀伤AML干细胞的新机制，为临床应用提供更充分的科学依据。

Acute myeloid leukemia is the most common hematopoietic malignancy in adults. Although treatment options are developing very fast with complete remission rate being nearly 70%, relapse remains to be the key reason for treatment failure. It has been demonstrated that leukemia stem cells (LSC) are quiescent, more resistant to chemotherapy and the basis for relapse after initial response. And thus targeting LSC is the most effective treatment option for AML. The survival of LSC is affected by both intrinsic and extrinsic components. Reactive oxygen species(ROS) and NF-κB are well-known intrinsic components while hypoxia inducible factor-1α(HIF-1α) which has cross-talk with NF-κB and its downstream targets such as stromal derived factor-1(SDF-1) , CXC receptor4 (CXCR4) and very late antigen-4(VLA-4) are important extrinsic components affecting the retention, adhesion of LSC in bone marrow. We have already demonstrated that Disulfiram/Cu(DS/Cu) could eliminate AML LSC through up regulation of ROS and down regulation of NF-κB. Moreover, in our preliminary experiments, we found that DS/Cu could down regulated HIF-1α of bone marrow stromal cells(BMSC) and AML LSC without affecting the survival of BMSC. Here, we hypothesize that DS/Cu can eliminate AML LSC by targeting both intrinsic and extrinsic pathways through up regulation of ROS and down regulation of HIF-1α and NF-κB..In this study, we will test these hypothesis through two objectives: (1) To determine that DS/Cu can affect the supportive function of BMSC to LSC through down regulation of HIF-1α,SDF-1 of BMSC and VLA-4 of LSC. (2) To determine the function of HIF-1αpathway in the elimination of AML LSC by DS/Cu and its relationship with ROS,NF-ΚB and multi drug resistance genes. .More specifically, we will use the following experimental techniques: (1) Screening electronic microscope, flow cytometry, colony formation and adhesion experiment will be used to test the survival rate , the proliferation and adhesion function of AML LSC in BMSC and LSC co-culture models. (2)HIF-1α,CXCR4, SDF-1,VLA-4 of AML LSC NOD/SCID mice model will be evaluated using immunohistochemistry method. (3) Laser scanning confocal microscope and flow cytometry will be used to determine ROS level of AML LSC. (4) Electrophoretic mobility shift assay, western blotting method will be used to test the expression of HIF-1α,CXCR4, SDF-1,VLA-4, NF-κB. (5)Western blotting and RT-PCR will be used to detect the level of multi drug resistance gene MDR1 and MRP1. Results from this study will lay the foundation for future development of a treatment option for acute myeloid leukemia.