手霉素调控白血病干细胞表达免疫激活物的作用和机制研究

Leukemic stem cells (LSCs) which are double resistant to chemotherapy and immunotherapy play the key role in the relapse of acute myeloid leukemia (AML) and highlight the critical need to eradicate the LSCs population. We already established LSC cell model which was resistant to chemotherapy and NK-mediated cytotoxicity. Down-regulation of NKG2D ligands were involved in protecting LSCs from NK-mediated attack. Furthermore, we found that up-regulating NKG2D ligands by manumycin increased the sensitivity of LSCs to NK cell-mediated killing. However，the exact effect and intrinsic mechanisms of manumycin on LSCs are not clear. This project will detect the effect of manumycin on viability, proliferation and apoptosis of LSCs and investigate the regulation of manumycin on LSCs immunoactivators including NKG2D ligand, DNAM-1 ligands and Ras、PI3K/AKT/ROS、NF-κB by flow cytometry, Soft agar clonogenic assay, western blot, real-time PCR, siRNA, and NOD/SCID mouse xenotransplant model with our LSC cellular model and LSCs isolated from relapsed AML patients. This study will confirm the effects and mechanisms of manumycin on regulation of immunoactivators expression in leukemic stem cell. This proposed research will allow us to identify new novel therapeutic targets for enhanced sensitivity of LSCs to NK cell-mediated killing.

化疗及免疫治疗双抵抗白血病干细胞（LSC）是急性髓性白血病(AML)复发的关键，清除LSCs至关重要。我们已成功构建LSC细胞模型；证实其具有抵抗化疗和免疫治疗的特性且免疫抵抗与LSCs低表达NKG2D配体相关；同时发现手霉素可上调LSC的NKG2D配体表达,提高其激发allo-NK细胞杀伤的活性。但迄今手霉素对LSCs的确切作用和机制尚未阐明。本项目拟利用自建的LSC模型和分选自复发AML病人的LSCs，采用克隆形成实验、流式细胞术、免疫印迹法、QPCR、 RNA干扰和荷LSC NOD/SCID小鼠等方法，以手霉素为干预手段，检测LSCs细胞活性、增殖及凋亡的变化，探讨对LSC表达表面免疫激活物（NKG2D和DNAM-1配体）和Ras、PI3K/AKT/ROS、NF-kB的影响。旨在阐明手霉素调控LSCs表达免疫激活物的作用及其机制，为有效激活allo-NK细胞清除LSCs提供新靶点。

化疗及免疫治疗双抵抗白血病干细胞（LSC）是急性髓性白血病(AML)复发的关键，清除LSCs至关重要。我们已成功构建LSC细胞模型；证实其具有抵抗化疗和免疫治疗的特性且免疫抵抗与LSCs低表达NKG2D配体相关；同时发现手霉素可上调LSC的NKG2D配体表达,提高其激发allo-NK细胞杀伤的活性。但迄今手霉素对LSCs的确切作用和机制尚未阐明。本项目利用自建的LSC模型和分选自复发AML病人的LSCs，采用克隆形成实验、流式细胞术、免疫印迹法、QPCR、 RNA干扰和荷LSC NOD/SCID小鼠等方法，以手霉素为干预手段，检测LSCs细胞活性、增殖及凋亡的变化，探讨对LSC表达表面免疫激活物（NKG2D和DNAM-1配体）和Ras、PI3K/AKT/ROS、NF-kB的影响。旨在阐明手霉素调控LSCs表达免疫激活物的作用及其机制，为有效激活allo-NK细胞清除LSCs提供新靶点。