双黄升白颗粒对肿瘤化疗骨髓抑制期造血干细胞微环境Wnt、Notch信号通路的调控

Myelosuppression is a common complication of chemotherapy of cancer, although colony-stimulating factor (CSF) is used to stimulate the bone marrow hematopoietic stem cells (HSCs) proliferation clinically, the CSF may promote tumor growth. Shuanghuang Shengbai(SHSB) granule is based on the tonifying kidney to regenerate bone marrow theory, which can promote the proliferation of HSCs and inhibit tumor growth. .HSCs micro-environment signaling pathway has an important role in the process of HSCs proliferation and is closely related to tumor growth. This study is on the basis of more than 20 years research to observe the effect of SHSB by using the model of tumor-bearing mice with chemotherapy induced myelosuppression with intraperitoneal injection, testing the blood routine, bone marrow nucleated cells and tumor size to verify the efficacy of SHSB; then separating the HSCs and counting, verifying its effect on proliferation of HSCs. Detecting gene expression of Wnt and Notch signaling pathway in bone marrow (main control signaling of HSCs micro-environment) to clearify the drug effects on signal transduction of HSCs micro-environment. Observing the same signaling pathways in tumor to analyze the safety of the drug. Through these experiments to make analysis of the regulation mechanism of SHSB on HSCs micro-environment. The study is to provide a theoretical basis to the transformation from empirical formula to clinical drug, to disscuss " tonifying kidney to regenerate bone marrow" theory from a new perspective.

骨髓抑制是肿瘤化疗常见并发症，临床常用集落刺激因子(CSF)促进造血干细胞(HSCs)增殖治疗，但CSF有促进肿瘤生长可能。双黄升白颗粒以补肾生髓为主，能在促进HSC增殖同时抑制肿瘤生长。.HSCs微环境信号通路对HSCs增殖有重要作用，且与肿瘤生长关系密切。本项目拟在20余年研究基础上，以荷瘤鼠注射环磷酰胺制成肿瘤化疗骨髓抑制模型，用双黄升白颗粒治疗。检测血常规、肿瘤大小，验证其疗效；分离计数HSCs，验证其对HSCs增殖的影响；观察超微结构，明确其对HSCs微环境结构的影响；检测骨髓Wnt、Notch信号通路(HSCs微环境主要调控信号)所包含主要基因的表达，明确其对HSCs微环境信号通路的影响；同时，对肿瘤中相同信号通路进行观察，分析其安全性；从而深入解析双黄升白颗粒治疗肿瘤化疗骨髓抑制期的作用机制，为经验方药向临床新药转化提供理论依据，并从新的角度阐述补肾生髓理论内涵。

背景：骨髓抑制是肿瘤化疗常见并发症，前期研究显示补肾生髓为主的双黄升白颗粒以能在促进骨髓造血干细胞(HSCs)增殖同时抑制肿瘤生长，其作用机制可能与Wnt、Notch信号通路有关。.方法：采Lewis肺癌荷瘤鼠腹腔注射环磷酰胺造成化疗骨髓抑制模型，用双黄升白颗粒干预治疗，常规计数白细胞、红细胞、血小板数量和肿瘤质量，流式细胞法检测骨髓HSCs率，FQ-PCR法及Western-blot法检测Wnt信号通路主要基因(Wnt、β-catenin、Frizzted、DSH、GSK3)与Notch信号通路主要基因(Notch1、Notch2、Notch3、Jagged、CSL )在骨髓与肿瘤中的表达。.结果：(1)疗效：治疗组白细胞数量、HSCs率、骨髓有核细胞数均高于模型组(P＜0.05)；各组红细胞、血小板、肿瘤质量均未发现明显差异(P>0.05)。(2)Wnt信号：治疗组Wnt、β-catenin、Frizzted、DSH、GSK3 mRNA及蛋白在骨髓中的表达高于模型组(P＜0.05)；Wnt、β-catenin、Frizzted、DSH mRNA在肿瘤中的表达低于模型组(P＜0.05)；各组GSK3 mRNA在肿瘤中的表达未发现差异(P>0.05)；治疗组Wnt蛋白在肿瘤中表达降低(P＜0.05)；治疗组β-catenin、Frizzted、DSH、GSK3蛋白在肿瘤中表达未发现差异(P>0.05)。(3)Notch信号：治疗组Notch1、Notch2、Notch3、CSLmRNA在骨髓中的表达高于模型组(P＜0.05)，在肿瘤中的表达则低于模型组(P＜0.05)；各组Jagged mRNA及蛋白的表达未发现差异(P>0.05)；治疗组Notch1、Notch2、Notch3、CSL蛋白在骨髓中的表达高于模型组(P＜0.05)；治疗组Notch1、Notch2、Notch3在肿瘤中表达降低(P＜0.05)；CSL、Jagged蛋白在肿瘤中表达未见差异(P>0.05)。.结论：双黄升白颗粒(1)可促进骨髓HSCs增殖；(2)可促进骨髓中Wnt、Notch信号通路中主要基因的表达；(3)对化疗骨髓抑制期肿瘤Wnt、Notch信号通路具有一定抑制作用；(4)用于肿瘤化疗骨髓抑制安全有效，具有目前临床常用药物所不具有的优势，有较好的新药开发价值。