白血病化疗的毒副反应及其耐药性仍是尚未解决的难题，研究安全有效抗白血病的中药新药很有必要。采用抑制增殖、诱导分化和促进凋亡试验，从20余个人参皂苷分离物中反复筛选出抗白血病作用最理想的低极性人参皂苷ALK。发明专利已授权，制备工艺稳定，成分含量明确，主含F4、Rh4、Rg3和Rk3等6个单体，总量75％。研究证实ALK体外抑制原代及多种白血病细胞株增殖，且诱导分化作用明显，体内治疗荷瘤和浸润白血病裸鼠的疗效显著。急性和长毒试验表明毒性很小，提示作用机制不同于细胞毒药物。鉴于白血病的发生与表观遗传调控异常密切相关，本项目拟开展ALK较全面的表观遗传学研究，包括DNA甲基化谱，组蛋白H3K9和H3K27乙酰化及miRNA表达谱，这三种机制存在相互作用，综合分析并确定共同调控的靶基因，即ALK调控白血病增殖分化的关键基因，为中医药学研究提供新技术，为研发安全有效抗白血病的中药新药提供实验依据。

Chemotherapy is the main therapeutic method for myeloid leukemia, but cytotoxicity drugs remains suboptimal because of serious side effects and drug resistance, which is still unresolved problem, it is necessary to search for both safe and effective drugs from Chinese herbs for treatment of myeloid leukemia. In our previously study, we approved that total saponins panax of ginseng (total ginsenosides) was effective for anti-leukemia by biological activity assay on proliferation, differentiation and apoptosis in several leukemia cell lines. In order to search for the effective compounds of anti-leukemia within total ginsenosides, we obtained more than twenty compounds isolated from total ginsenosides, each of them contained several monomers. Finally, we confirmed that low polarity of panax ginsenosides ALK was the most effective for anti-leukemia among more than twenty compounds though biological active assay of leukemic cells above，and its invention patent applied has been authorized. Now, pharmacological preparation techniques of ALK was stable, its specific elements was mainly composed of six monomers F4、Rh4、Rg3 and Rk3 etc. The total quantity was 75% in purification analyzed by chromatogram - mass spectrum and HPLC. Our study showed that ALK was effective to not only inhibit proliferation of both patients' leukemic cells and cell lines, but also induce their differentiation by cytological experiment and animal models of leukemia in both vitro and vivo. Furthermore, both acute and chronic toxicological tests showed the toxin of ALK was very lower. All of results above indicated that anti-leukemia mechanism of ALK was different from the cytotoxicity chemotherapeutic drugs. Since the epigenetics regulation is closely related to occurrence and development of leukemia, in this project we will focus on the epigenetic regulation mechanism of leukemia cells induced by ALK, including DNA methylation profile, genomic context histone H3K9 and H3K27 acetylation patterns, as well as miRNA chip expression profile, which the roles of three mechanisms will be interacted each other, so as to clarify the mechanism of ALK, and find common target genes and proteins by comprehensive analysis of three results in ALK treated leukemic cells. The new methods and techniques can be established in this project for investigation related Chinese medicines of anti-leukemia or tumors. It also provided the experimental evidence to develop ALK as a new Chinese patent drug for effectively treating leukemia without obvious side efficacy.

白血病化疗药物的毒副反应及其耐药性仍是尚未解决的难题，研究安全有效抗白血病的中药新药很有必要。创新中药低级性人参皂苷ALK体内外抑制白血病细胞增殖和诱导分化作用显著，发明专利已授权。本项目通过较全面的表观遗传学研究，揭示ALK调控白血病增殖分化的关键靶向基因和蛋白。主要研究和重要结果：①ALK有效地诱导红系K562、HEL、单核系SHI-1、巨核系Meg-01等多种白血病细胞分化，并阻滞细胞增殖周期。②通过调控DNA甲基化，上调抑癌基因、DNA修复和分化相关基因，下调致癌基因而发挥抗白血病作用。③ALK靶向上调白血病特异性WT1基因的甲基化，高甲基化WT1使其mRNA和蛋白表达下调。④分析ALK诱导的miRNA表达谱，筛选并验证了白血病或肿瘤相关差异10倍以上的miRNA。⑤ALK靶向上调白血病密切相关的miR-148b-5p，通过转染细胞过表达，验证其具有诱导分化和阻滞细胞周期的作用。⑥计划外研究结果：分离出ALK主含的5个单体，分别测定抗白血病的生物学活性，筛选并确定Rh4、Rk3是抗白血病的有效成分。⑦Rk3诱导K562细胞乙酰化上调主要9个组蛋白为H2BA、H4A和H2AJ等，以上调H2乙酰化为主，与诱导白血病细胞分化的趋势呈正相关。⑧计划外研究结果：ALK具有逆转K562/ADM白血病细胞耐药的作用。科学意义：①从表观遗传水平证明ALK抗白血病的有效性，特色优势是通过表观遗传调控而诱导白血病细胞分化及阻滞周期。②建立适合中医药研究的新技术和新方法。③为开发安全有效治疗白血病的中药新药提供实验依据，上述结果将作为新药的部分药效学资料，用于上报国家药监局，申请中药新药临床试验批件。④相关研究发表论文6篇，均标注基金会资助，其中SCI收录2篇。培养在职人员晋升副高2名，博士毕业1名、硕士毕业4名。承办小型国际会议1个，承办全国会议1个；全国会议学术报告2次。