基于磷脂爬行酶1（PLSCR1）分子靶点的汉黄芩苷抗人急性髓系白血病（AML）的作用机制研究

PLSCR1 is a lipid-binding protein that has been proposed to accelerate redistribution of plasma membrane phospholipids between the inner and outer leaflets. It also plays potential roles in hematopoiesis and leukemogenesis. Traditional chemical therapy shows toxic side effects and low remission rate in acute myeloid leukemia. Differentiation therapy has been reported to show less toxicology and side effects, therefore, new differentiation inductors are required to be discovered. It is reported that wogonin has strong effects of anti-leukemia in vitro, wogonoside is the main metabolites that play activity in vivo, our study shows that wogonoside not only induced the cycle arrest and differentiation of AML cell lines (HL-60,U937 cell) but also up-regulated expression of PLSCR1, at the same time, preliminary study showed that wogonoside had strong activity to AML primary cells both in vivo and in vitro. The differentiation induction effects of wogonoside in AML cell and regulation of PLSCR1 function have not been reported. This project aims to study the regulation process that under the action of wogonoside, PLSCR1 activates IP3R1 promoter in nucleus after depalmitoylation, thus affecting the function of calcium ion as well as the mechanism of action to AML primary cells in vivo and in vitro, at the same time, to provide new evidence for clarify the mechanism of wogonin in anti-leukemia and new approach to the study of treatment for leukemia with natural products.

PLSCR1参与白血病细胞分化的过程，是目前分化诱导疗法治疗AML的研究热点。传统化疗药物应用于AML治疗，副作用大，缓解率低，而分化诱导疗法药物副作用明显减少，因此新的治疗AML的分化诱导剂亟待开发。文献报道汉黄芩素具有较强的体外抗白血病作用，汉黄芩苷是汉黄芩素的体内主要活性代谢产物，有关汉黄芩苷诱导人AML细胞分化的作用国内外文献未见报道。本课题组初步研究表明，在AML细胞系中，汉黄芩苷能诱导细胞发生周期阻滞和分化及上调PLSCR1的表达，并能抑制裸鼠移植瘤的生长；在人AML原代细胞中，汉黄芩苷能诱导细胞分化并延长原代细胞接种NOD/SCID小鼠的生存期。本项目旨在深入探讨汉黄芩苷抗AML作用机制，并研究它对AML细胞系及人原代细胞中PLSCR1去棕榈酰化后入核激活IP3R1启动子，从而影响钙离子功能的调控过程，为阐明其抗AML的确切机制提供新依据，同时为天然产物治疗白血病提供新思路。

PLSCR1 参与白血病细胞分化的过程，是目前分化诱导疗法治疗AML的靶点之一。传统化疗药物应用于AML 治疗，副作用大，缓解率低，而分化诱导疗法的药物副作用明显减少，因此新的治疗AML 的分化诱导剂亟待开发。文献报道汉黄芩素具有较强的体外抗白血病作用，汉黄芩苷是汉黄芩素的体内主要活性代谢产物，有关汉黄芩苷诱导人AML 细胞分化的作用国内外文献未见报道。本项目旨在进一步探讨汉黄芩苷抗AML 的作用机制，研究它对AML 细胞系及人原代细胞中PLSCR1去棕榈酰化后入核激活IP3R1 启动子，从而影响钙离子功能的调控过程，为进一步阐明其抗AML 的确切机制提供新依据。本研究依据PLSCR1对汉黄芩苷的响应程度选取了三种类型的样本开展本课题的研究，阐明PLSCR1的核转位机制，确认汉黄芩苷诱导的PLSCR1去棕榈酰化是PLSCR1核转位的重要环节。同时利用PLSCR1 siRNA、IP3R1的抑制剂2-APB、外源Ca2+的螯合剂EGTA证实PLSCR1-IP3R1-Ca2+信号通路在汉黄芩苷诱导的人AML原代细胞分化中的关键作用。本项目首次发现了汉黄芩苷对人AML原代细胞中PLSCR1与N-RAS去棕榈酰化修饰的作用，以及阐明酰基蛋白硫酯酶APT1介导的PLSCR1/N-RAS去棕榈酰化和转位在汉黄芩苷诱导的人AML原代细胞分化及抗白血病中的重要作用。最后，本研究分别构建了U937细胞裸鼠移植瘤模型及不同类型人原代AML细胞接种的NOD/SCID小鼠模型来研究汉黄芩苷的体内抗AML作用。汉黄芩苷在U937细胞裸鼠移植瘤中显著上调PLSCR1及IP3R1的表达，同时影响分化与周期相关蛋白的表达。同样，汉黄芩苷上调AML原代细胞接种的NOD/SCID小鼠外周血CD45的表达，并显著降低骨髓中AML细胞的浸润及增加骨髓中AML细胞的PLSCR1和IP3R1表达。同时汉黄芩苷有效延长了接种AML原代细胞的NOD/SCID小鼠的生存期，并且改善小鼠生存状态。这些结果表明，汉黄芩苷在体内也具有着较好的抗AML作用，说明汉黄芩苷具有成为抗AML新药的潜力，为天然产物治疗白血病研究提供新思路。