基于线粒体氧化呼吸与能量代谢研究中医扶正与祛邪疗法治疗白血病的作用机制

In recent years, the research priorities of traditional Chinese medicine(TCM) treatment of hematological and neoplastic diseases are immune regulation, synergism and attenuation, apoptosis-inducing and so on. It is still blank that TCM treatment of malignant tumor by intervention of mitochondrial oxidative respiration and energy metabolism. Mitochondria provide 90% energy for cells and it is extremely important to maintain cellular functions. In previous studies, we had discovered Chinese herbal compound could affect expression of two kinds of mitochondria mediated apoptosis related factors(Bcl-2,caspase-3). In order to research TCM treatment of leukemia through mitochondrial pathway, we establish an animal model of acute myelogenous leukemia(AML) by KG-1a cells in NOD/SCID mice. We choose some experimental indexs which could reflect functions of mitochondrial oxidative respiration and energy metabolism, such as mitochondrial reactive oxygen species(ROS), mitochondrial membrane potential, activity of mitochondrial respiratory chain enzyme complexes, expression of mitochondrial ATPase mRNA and so on. According to the experimental data, we hope to clarify the mechanism of TCM Fuzheng and Quxie treatment of leukemia by intervention of mitochondrial oxidative respiration and energy metabolism. We take the study as an exploratory research for the frontier of TCM treatment of hematological malignancies through mitochondrial pathway. Guided by this theory, we could provide the basis for studying new anticancer chinese herbal medicine.

近年来中医药治疗白血病等血液肿瘤疾病的研究多针对免疫调节、增效减毒、诱导凋亡等方面,而中医药干预线粒体氧化呼吸与能量代谢治疗肿瘤的研究尚处空白。线粒体提供细胞90%的能量，在维持细胞功能方面处于重要地位。我们前期研究发现中药复方可影响白血病细胞线粒体凋亡相关因子Bcl-2、caspase-3表达。为进一步研究中医药通过线粒体途径治疗白血病的作用，拟采用KG-1a细胞株在NOD/SCID小鼠平台上建立人急性髓系白血病模型，选用能反映线粒体氧化呼吸与能量代谢功能的线粒体活性氧簇、线粒体膜电位、线粒体呼吸链酶复合体活性、线粒体ATP酶mRNA等指标，探索中医扶正与祛邪疗法干预线粒体氧化呼吸与能量代谢治疗白血病的作用机制，对中医药通过线粒体途径治疗血液肿瘤疾病这一新领域进行探索性研究，为在此理论指导下研究新的抗癌中药提供理论依据。

线粒体氧化呼吸与能量代谢在白血病细胞的增殖、分化与凋亡过程中起到重要的作用。中医扶正与祛邪疗法能够通过调控白血病发生发展的关键环节——线粒体氧化呼吸与能量代谢诱导白血病细胞凋亡。本项目应用KG-1a细胞以NOD/SCID小鼠为平台，成功建立人急性髓系白血病疾病模型，分别用益气养阴方中的扶正药物、祛邪药物和扶正祛邪药物对各组小鼠进行灌胃饲养，细胞周期及凋亡检测结果证实益气养阴方及其拆方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，其中益气养阴方组和祛邪组调节显著。透射电镜下可见益气养阴方干预后细胞核颜色加深，形态固缩，线粒体形态数量减少，嵴排列紊乱，线粒体结构损伤严重。免疫组化结果提示白血病小鼠通过各组治疗后，线粒体相关凋亡因子Cyt-C、Apaf-1、Smac/Diablo、AIF的表达显著升高，线粒体呼吸链酶复合体功能损伤，益气养阴方组的作用更强，推测各组通过线粒体的途径促使白血病细胞凋亡，从而起到治疗白血病的作用。DNA测序结果提示各组均有基因突变，但各组间差异不显著。Western Blotting结果提示益气养阴方及其拆方可以诱导KG-1a细胞线粒体介导的凋亡相关因子Cyt-C、AIF、Apaf-1、Smac/Diablo表达增高，益气养阴方及祛邪组干预KG-1a细胞后，线粒体膜电位降低，ROS释放增多，提示益气养阴方可以诱导白血病细胞的早期凋亡。本项目通过检测人白血病细胞线粒体氧化呼吸和能量代谢相关指标的变化，探索中医扶正与祛邪疗法干预线粒体氧化呼吸与能量代谢治疗白血病的作用机制，对中医药通过线粒体途径治疗血液肿瘤疾病这一新领域进行探索性研究，为在理论指导下研究新的抗癌中药提供了理论依据。