莱菔硫烷通过Sonic Hedgehog信号通路调控白血病干细胞增殖的机制研究

Leukemia stem cells (LSCs) are the main factors for drug resistance and relapse of leukemia. To cure leukemia, clearing LSCs is the only one way. It has been proved that Sonic hedgehog (Shh) signaling pathway is involved in the occurrence and development of varieties of malignan tumors. Our previous studies showed that Sulforaphane (SFN) can inhibit the self-renewal and proliferation of acute myeloid leukemia stem cell. SFN inhibits Shh expression in the Shh signaling pathway in the leukemia stem cells (CD123+). Thus, we speculate that SFN inhibits self-renewal and proliferation of Leukemia stem cells through the modulation of Shh signaling pathway. However, it is unclear how SFN regulates Shh signaling pathway in leukemia stem cells. In the present project, based on in vitro cell culture and animal model, firstly, we will analyze how SFN affects CD123+ cell proliferation of LSCs, and investigate the relationship between the activiation of of Shh signaling pathway and LSCs proliferation with FACS and RT-PCR methods and so on. Secondly, we will further study how SFN affect the activing expression of key molecules in the Shh signaling pathway, and reveal the function and mechanism that SFN regulates the Shh signaling pathway to inhibit the proliferation of LSCs. The results will lay the foundation that SFN becomes a new drug to improve the treatment of leukemia.

研究证明白血病干细胞（LSCs）是白血病耐药和复发的主要根源，只有清除LSCs才有可能根治白血病，已证实Sonic hedgehog（Shh）信号通路参与了多种恶性肿瘤发生、发展。我们研究发现莱菔硫烷（SFN）不仅可以抑制急性髓系白血病干细胞自我更新和增殖能力，且对Shh信号通路中的Shh蛋白的表达具有抑制作用。由此推测SFN可能通过调控Shh信号通路抑制白血病干细胞的自我更新和增殖。本项目中我们拟建立体外细胞培养和动物模型两种实验体系，采用流式细胞术、Western-bot、RT-PCR等方法首先分析SFN对白血病干细胞（CD123+）的增殖影响及机制；进一步剖析Shh信号通路的激活与白血病干细胞增殖的相关性；再研究SFN对Shh信号通路中关键分子的活性表达影响，阐明SFN通过调控Shh信号通路抑制白血病干细胞增殖的机制。为SFN成为辅助治疗白血病的新药提供理论基础和实验基础。

异常的Sonic Hedgehog（Shh）信号通路激活与多种肿瘤的发生有关。针对白血病中Shh信号通路的异常激活是一个很有希望的治疗靶点。莱菔硫烷（Sulforaphane, SFN）是一种从十字花科蔬菜中提取的异硫氰酸酯，对多种肿瘤具有良好的抗癌作用，但SFN是否影响急性白血病增殖尚未阐明。由于白血病干细胞的自我更新能力，急性白血病的复发率很高。本研究探讨了SFN对急性髓系白血病中白血病干细胞自我更新和增殖的影响及其相关分子机制。我们发现SFN在体外和体内都抑制了白血病干细胞的增殖和自我更新。同时，我们观察到SFN可以调节白血病细胞的干细胞特性。在SFN处理后，Shh信号通路中关键因子的表达在转录和蛋白质水平上显著降低。为了进一步明确Shh信号通路在SFN介导白血病干细胞增殖和自我更新能力中的分子机制，我们随后操纵白血病细胞中的Shh基因，使用慢病毒载体转导过度表达该基因及通过siRNA敲除该基因。结果表明，SFN对Shh过度表达细胞的增殖抑制作用大于Shh下调细胞，提示SFN抑制白血病干细胞增殖和自我更新与Shh信号通路密切相关。总之，这些结果表明SFN是一种有效的抗白血病药物，通过Shh信号通路调控白血病干细胞增殖和自我更新。