基于Wnt/β-catenin信号通路研究当归补血汤调控造血干细胞促进造血重建的机制及中医"气血相生"的机理

Bone marrow depression is the commonly adverse reaction of the most immunodepressant. The apoptosis of Bone marrow Hematopoietic Stem Cell, which is regulated directly by the Wnt/β-catenin signal pathway, is the chief factor to the hematopoietic reconstitution. Base on established bone marrow Hematopoietic Stem Cell apoptosis mice model and bone marrow depression model, and confirmed that Danggui Buxue Decoction can depress the apoptosis of mice bone marrow Hematopoietic Stem Cell and regulated Wnt and it's acceptor, we would like to use R Spondin-1、LiCl and Dickkopf-1、Frz protein for intervention, to test the expression of signal molecules gene、protein such as Wnt、Dsh、GSK3β、Axin、APC、β-catenin by the technology of RT-PCR and Western Blot. In order to study the signal transduction process of Wnt/β-catenin deeply and systematically, and identify specific function target point of Danggui Buxue Decoction and it's different composition on this transduction, meanwhile, clarify the mechanism of Danggui Buxue Decoction's role on the promote the realignment of hematopoiesis and the traditional chinese medicine theory of "Qi and Blood generate each other", provide more exactly methods for chinese medical prevention and treatment on bone marrow depression which is caused by immunodepressant, and offer basis for traditional chinese medicine theory of "Qi and Blood generate each other".

骨髓抑制是大多数免疫抑制剂共有的不良反应，骨髓造血干细胞的过度凋亡是影响造血重建的主要因素，而Wnt/β-catenin信号通路直接调控造血干细胞的凋亡。.本课题组在建立小鼠骨髓造血干细胞凋亡、骨髓抑制模型及证实当归补血汤能抑制小鼠骨髓造血干细胞凋亡、调控Wnt及其受体表达的基础上，通过R Spondin-1、LiCl和Dickkopf-1、Frz蛋白的干预，采用定量RT-PCR、Western Blot等技术，检测Wnt及其下游Dsh、GSK3β、Axin、APC、β-catenin信号分子基因、蛋白的表达，深入研究骨髓造血干细胞内Wnt/β-catenin信号转导过程，观察当归补血汤及其拆方（益气法、补血法）调控该信号转导通路的作用靶点，阐明当归补血汤介导该信号通路促进造血重建的机制及中医"气血相生"的分子机理，为中医药防治骨髓抑制提供更确切的方法，为中医"气血相生"理论提供实验依据。

骨髓抑制是大多数免疫抑制剂共有的不良反应，当归补血汤具有防治骨髓抑制的作用，但其机理尚不明确。本项目从Wnt/β-catenin信号通路的角度探讨当归补血汤调控造血干细胞的作用机制。提出免疫抑制剂导致的骨髓抑制属于中医“血虚”证候，治疗应以“补血为主、补气生血”为法促进造血重建的观点。建立了可重复的骨髓抑制小鼠模型。明确了当归补血汤治疗骨髓抑制的确切疗效。发现了当归补血汤具有提高骨髓Wnt3a蛋白及其受体表达水平的作用。证实了当归补血汤能提高骨髓造血干细胞中Frizzled 2、Dvl3、β-catenin mRNA，并降低p-GSK3β、APC mRNA表达水平。体外研究发现，当归补血汤可通过提高Dvl3 mRNA、降低p-GSK3β mRNA的表达而影响Wnt信号转导通路。此外，本项目同时进行了当归补血汤拆方研究，从分子水平证实了当归补血汤中任何单味成分（黄芪、当归）在提高骨髓造血干细胞Frizzled 2、Dvl3、β-catenin mRNA表达水平方面，或者在降低p-GSK3β、APC mRNA表达水平方面，它们的作用均差于二者的组合物——当归补血汤。本项目为骨髓抑制研究提供了一种合适的骨髓抑制小鼠模型，为中医药防治骨髓抑制提供更确切的方法和科学依据，为中医“气血相生”理论提供实验依据。该研究已完成论文9篇，已发表3篇（1篇SCI，IF3.6分）；申请国家发明专利1项。