基于《金匮》“阴阳毒病”证治规律研究升麻鳖甲汤对NOD/SCID白血病模型线粒体动力学的干预作用

Mitochondria are the most important organelles in eukaryotic cells, and are closely related to tumorigenesis。Changes in the balance of mitochondrial dynamics can determine the fate of tumor cells through the mitochondrial autophagy and apoptosis pathways, so mitochondrial dynamics regulation is a key link in the regulation of tumor mitochondria.In my doctoral project, the important role of mitochondrial dynamic imbalance in the pathogenesis of acute leukemia, especially the relapsing refractory leukemia, was clarified for the first time.On the basis of the previous research of my doctor's project, this study will take relatively mature NOD/SCID animal model of leukemia as the research object, and used shengmabiejia decoction with remarkable clinical efficacy to intervene in the treatment of leukemia，and study on the effect of mitochondrial dynamics under the intervention of shengmabiejia decoction based on the syndrome and treatment rules of "yin-yang toxicosis" in synopsis of the golden chamber,we explore to illuminate the guiding significance of traditional Chinese medicine theory to the diagnosis and treatment of malignant blood system diseases,provide new ideas for the Chinese medicine diagnosis and treatment in acute leukemia under the guidance of the traditional theory, modern cell and molecular biology.

线粒体是真核生物细胞最重要的细胞器，与肿瘤发生关系密切。线粒体动力学平衡的改变能够通过线粒体自噬与凋亡途径决定肿瘤细胞的命运，因此线粒体动力学调控是肿瘤线粒体调控的关键环节，在课题负责人的博士课题中首次明确了线粒体动力学失衡在急性白血病尤其是复发难治急性白血病发病中的重要作用。本研究是在博士课题前期研究基础上，以较为成熟的NOD/SCID急性白血病模型为研究对象，以临床上治疗白血病疗效显著、单味中药或有效成分抗白血病效应明确的升麻鳖甲汤进行干预，并依据《金匮要略》中“阴阳毒病”证治规律对其进行拆方研究，探讨该方对于线粒体调控的关键环节-线粒体动力学的干预作用，以进一步阐明中医传统理论对急性白血病诊治的指导意义，为中医药传统理论结合细胞和分子生物学诊治急性白血病提供新的思路。

急性白血病（Acute Leukemia,AL）是起源于造血干细胞（HSCs）的肿瘤性疾病，严重危害人类健康。中医药能够通过调控白血病发生发展的关键环节--线粒体动力学诱导白血病细胞自噬与凋亡。本项目应用KG-1a细胞以NOD/SCID小鼠为平台，建立人急性白血病疾病模型，分别用升麻鳖甲汤及其拆方药物对各组小鼠进行干预。NOD/SCID-AL小鼠经升麻鳖甲汤全方及拆方治疗后，线粒体相关凋亡因子Bax的表达显著升高，Mcl-1显著降低，提示升麻鳖甲汤全方及拆方均具有诱导白血病细胞凋亡的作用。细胞周期及凋亡检测结果证实升麻鳖甲汤可以使白血病细胞停滞于G1期，无法进入S期，抑制细胞的增殖，细胞凋亡率明显升高。透射电镜下可见NOD/SCID-AL模型组小鼠骨髓中出现线粒体形态呈卵圆形，细胞核形状不规则,线粒体增多，体积增大，嵴轻微扩张，提示线粒体处于融合状态。经升麻鳖甲汤全方及拆方治疗后小鼠骨髓中可见大量溶酶体与自噬体，线粒体体积减小，嵴欠清晰且排列较不整齐，少数可见嵴轻度溶解，基质不均。提示升麻鳖甲汤及拆方可改变白血病小鼠骨髓中线粒体形态学、破坏线粒体超微结构，引起线粒体分裂，诱导白血病细胞自噬与凋亡。免疫荧光结果显示经升麻鳖甲汤全方及拆方治疗后小鼠骨髓中线粒体融合相关因子Mfn2、Opa1表达明显降低，线粒体分裂相关因子Fis-1以及自噬相关因子Parkin表达明显升高。Western Blotting结果提示升麻鳖甲汤可调节白血病小鼠骨髓中线粒体融合/分裂平衡，并促进白血病细胞自噬，诱导白血病细胞凋亡。本项目通过检测人白血病细胞线粒体融合、分裂、自噬相关指标的变化，探索以“阴阳毒”理论为指导的升麻鳖甲汤干预线粒体动力学治疗白血病的作用机制，为中医药治疗急性白血病提供了可靠的理论依据。