健脾补肾方干预辐射损伤小鼠wnt细胞信号通路的表达

Chinese medicine believes that the invigorating spleen and kidney is close to the body's hematopoietic and immune function.This law is the rule to formate the invigorating spleen and kidney preciption,the preciption has a protective effect on the radiation damage,pre-clinical trials showed that in order to improve the peripheral blood and enhancedimmune function,early animal experiments to generate stem cells from bone marrow,bone marrow and spleen related apoptosis gene expression and the spleen of thehistopathological changes described the mechanism of injury protection effect in kidney and spleen prescription on the mouse radiation.Wnt signaling pathway has been foundtranslation abnormalities in the study of the blood system disease,we can see the expression of some of the abnormal signal in the cell signaling pathways can lead to abnormal hematopoiesis.Research group consider based on previous work,and intend to use PCR and westerm methods to detect the expression of abnormal signal of Wnt signaling pathway of bone marrow sten cells,and clear the mechanisms of protective effect on invigorating spleen and kidney prescription to chemical injury from the angle of the cell signaling,in order to provide theoretical basis for new drug discovery.

中医认为"健脾补肾"与机体的造血和免疫功能关系密切。以此为法则组建"健脾补肾方"，该方对辐射损伤具有保护作用，前期临床试验表明该方能提高外周血象及增强免疫功能，前期动物实验从骨髓干细胞的生成、骨髓及脾脏相关凋亡基因表达及脾脏组织病理学改变等方面阐述了补肾健脾方对小鼠辐射损伤保护作用的机制。在血液系统疾病的研究中已发现Wnt信号通路存在翻译异常，可知细胞信号通路的某些异常信号表达可导致造血异常。本课题组考虑在前期工作基础上，拟采用PCR及western方法检测骨髓干细胞Wnt信号通路的异常信号表达，从细胞信号转导角度阐明健脾补肾方对化学损伤保护作用的机制，为新药研发提供理论依据。

“健脾补肾方干预辐射损伤小鼠wnt信号通路的表达”，将由炙甘草、熟地黄、黄芪、陈皮、泽泻组成的健脾补肾方应用于直线加速器全身照射所致的辐射损伤小鼠模型，通过基因组芯片检测、病理观察、western-blot、RT- PCR、细胞培养等技术方法，将形态改变与功能变化相结合，观察健脾补肾方对辐射造血功能的影响。结果表明：1、 差异基因表达谱分析显示，辐射损伤后，小鼠骨髓细胞基因表达谱发生显著变化，辐射组共筛选到261个显著性差异表达基因，其中上调基因108个，下调基因153个；与辐射组比较，健脾补肾方大、小剂量组干预后，分别使辐射诱导的差异表达基因中35个、42个基因表达发生逆转；富集的GO功能分析显示，这些发生逆转的差异基因主要涉及细胞黏附、物质转运、细胞生长发育、增殖分化，信号转导、物质代谢、免疫与刺激等多个方面；富集的Pathway分析显示，有34个差异表达基因在造血相关的10条通路富集，其中以Wnt信号通路中差异基因表达最为丰富。2、与正常组相比，模型组小鼠骨髓Wnt3a基因及蛋白、β-catenin蛋白、TCF蛋白、CyclinD1基因表达量明显下降，粒-巨细胞集落数减少，PPAR-γ蛋白表达量增加，骨髓脂肪空泡面积增加；与模型组比较，药物干预后（健脾补肾方大剂量组、小剂量组及阿胶浆组），Wnt3a蛋白、β-catenin蛋白、TCF蛋白表达量及粒-巨细胞集落数均升高，PPAR-γ蛋白表达量、骨髓脂肪空泡面积明显减少。3、综合评价不同剂量的健脾补肾方和复方阿胶对小鼠辐射损伤模型的预防和治疗作用，以健脾补肾方小剂量组疗效最佳，大剂量组次之。本实验条件下，小剂量健脾补肾方防止辐射损伤效果是较理想的。综合上述研究结果，已在国内外期刊上发表论文4篇。