定清片在局部真实世界环境下抗击白血病复发机制研究

The relapse of leukemia is rooted in the existing of leukemia stem cell. As the evolution of the leukemia cells follow the general rule of biological evolution, and existing treatment methods are difficult to completely eradicate leukemic stem cells and (or) former leukemia stem cells, our research group proposed the thesis that the complete eradication of leukemic stem cells is neither realistic nor necessary. Hence, the prevention and treatment of leukemia relapse should follow the guidance of the TCM theory of "strengthening the right and expelling the evil" and pay full attention to stimulating the disease-resistant and rehabilitation ability of the human body itself. We can inhibit the excessive proliferation of leukemic stem cells by maintaining the number and function of normal hematopoietic cells, and fight against leukemia stem cells with various methods timely and properly. In order to verify the above hypothesis, we will conduct the research in the light of "real world study" concept and following the paradigm developed by Chenzhu's research group in their study of treating acute promyelocytic leukemia with Compound Realgar and Natural Indigo Tablet. We will study in vitro the molecular biological mechanism of Dingqing Tablet, a compound preparation of Chinese materia medica whose validation has been verified by more than 20 years of clinical application, and the study may also demonstrate the scientific connotation of the TCM theory of "strengthening the right and expelling the evil". The observational index we selected, including BCL-2 gene oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells,and Growth Factor Independence 1 (Gfi1) in Lymphoblastic Leukemia, were announced this year (2013) or in recent years and are cutting-edge experimental technique.

白血病复发的根源在于存在白血病干细胞。鉴于白血病细胞的演化遵循生物进化的一般法则，同时现有治疗方法难以完全根除白血病干细胞和（或）前白血病干细胞，据此课题组提出"彻底根除白血病干细胞既不现实亦无必要"的论断，继而认为防治白血病复发可以以中医"扶正祛邪"理论为指导，通过维持正常造血细胞的数量与功能来抑制白血病干细胞的过度增殖，并适时采用各种方法对白血病干细胞予以适度打击。为了验证上述假说，课题组遵循"真实世界研究"理念，以陈竺研究组关于复方黄黛片治疗急性早幼粒细胞白血病的研究成果为范例，通过体外研究经过20余年临床验证的有效中药复方--定清片--抗击白血病复发的系统分子生物学机理，以阐释中医"扶正祛邪"理论的科学内涵。所选择的观察指标，包括白血病干细胞氧化磷酸化能量代谢调控基因BCL-2和急性白血病存活依赖蛋白Gfi1等，均于今年（2013年）或近年公布，具有国际前沿性。

目的：本研究旨在探讨定清片抗击白血病复发可能的分子生物学机理，为临床治疗白血病提供理论依据。方法：收集完全缓解急性白血病患者60例、未缓解急性白血病患者30例、非血液肿瘤患者30例，采用功效拆方及全骨髓液培养的方法，将定清片全方及其“祛邪”、“扶正”拆方组药物体外作用于各组患者骨髓细胞，并运用显微镜、流式细胞术、实时荧光定量PCR（Real-time PCR）、基因测序、Western blot等方法，比较药物干预前后骨髓原始细胞数、造血干细胞数、MRD阈值、FLT3突变基因、TET2突变基因、BCL-2基因、p53基因及Gfi1蛋白表达的变化。结果：（1）未缓解组患者骨髓原始细胞数显著高于完全缓解组和非血液肿瘤组（P<0.001）；经定清片全方组、祛邪组和扶正组药物干预后，患者骨髓原始细胞数较药物干预前及空白组下降，其中定清片全方组原始细胞数下降最为明显，祛邪组次之，扶正组最弱。（2）未缓解组患者CD34+细胞及MRD值>0.01%显著高于完全缓解组和非血液肿瘤组（P<0.001）；经定清片全方组、祛邪组和扶正组药物干预后，患者CD34+细胞及MRD值>0.01%人数较药物干预前及空白组减少，其中定清片全方组和祛邪组减少更为显著（P<0.001）。（3）未缓解组患者FLT3、TET2、BCL-2及p53基因突变阳性发生率分别为30%、16.7%、20%和13.3%，均高于完全缓解组和非血液肿瘤组患者（P<0.05）；而经定清片全方组、祛邪组及扶正组药物干预后各基因突变位点数与干预前及空白组比较，差异无统计学意义（P>0.05）。（4）未缓解组患者Gfi1蛋白水平显著高于完全缓解组和非血液肿瘤组（P<0.05）；经定清片全方组、祛邪组和扶正组药物干预后，患者Gfi1蛋白水平较药物干预前及空白组下调，其中定清片和祛邪组表现出的下调作用最明显（P<0.05）。结论：（1）FLT3、TET2、BCL-2及p53基因突变与白血病的发生关系密切，但定清片并不能降低各基因的突变位点数目。（2）定清片可降低白血病患者骨髓原始细胞数、CD34+细胞数、MRD以及下调Gfi1蛋白表达水平，推测这可能是其抗击急性白血病复发的部分作用机制。