复方扶方藤合剂对体外模拟大鼠造血龛造血调控机制研究

In the body,when the Spleen qi is deficient,the transformation of food into blood and qi is impaired and the blood is unable to nourish the Heart.Compound Fufangteng Mixture can augment the qi and tonify the blood ,strengthen the Spleen and nourish the Heart. In clinic,Compound Fufangteng Mixture is used for an adjuvant therapy to increase hemocytes for patients in blood diseases.The latest study indicates that it is mesenchymal stem cells and hematopoietic stem cells form a unique bone marrow niche. It was found in our previous studies that Compound Fufangteng Mixture could increase hematopoietic stem cell(HSC) CFU-Mix（colony-forming unite-mix）in peripheral blood in mouse ,and it could promote the proliferation activities of rat's bone marrow mesenchymal stem cells significantly. Compound Fufangteng Mixture can activate the Notch and Wnt signal pathway, their key genes and expressed protein in Notch and Wnt signal pathway of mesenchymal stem cells (MSCs) in rat were up-regulated. However, there is no any report for Compound Fufangteng Mixture whether it has effect on hematopoietic stem cell niche in rat at home and abroad. We intend to study the effection of Compound Fufangteng Mixture on hematopoietic stem cells niche in rat. In this study, the whole bone marrow adherent method would be used to isolate, culture and purify the mesenchymal stem cells,and immunomagnetic bead method would be used to isolate and purify hematopoietic stem cells, then identified by flow cytometry.Bone marrow mesenchymal stem cells (MSCs) in co-culture with hematopoietic stem cell to construct a two-demensional culture system mimicking niche in vitro.Mimicking niche would be treated with Compound Fufangteng Mixture . Befor and after treating ,using cDNA microarray to compare the differences of gene expression profile of MSCs and HSC in niche; and using antibody microarray to compare the differences of cytokine profile in supernatants in niche. The differentially expressed genes and cytokines were selected, for the quantitative determination of differentially expressed genes and cytokines with qPCR and ELISA; Analysis transcription regulation signal pathway net by differentially expressed genes. To investigate the effect of Compound Fufangteng Mixture on mimicking niche, studying the mechanism of the effection of Compound Fufangteng Mixture on mimicking niche.Annotating Chinese medical theory "The Spleen is the source of qi and blood formation" with studying results. Supplying theory for supporting co-used Chinese Herbal Formulas with MSCs and HSC co- transplantation in clinic.

复方扶芳藤合剂具有益气健脾，补血养心的作用,临床上血液病患者常将其作为辅助提升血细胞的药物。前期工作发现复方扶芳藤合剂对大小鼠间充质干细胞（MSCs）及造血干细胞（HSC）有较好的促增殖及调控作用，研究证明MSCs与HSC构成造血龛，因此我们推测复方扶芳藤合剂对造血龛造血有调控作用。为证实假说，本研究拟以大鼠MSCs与HSC建立二维培养体系，体外模拟造血龛，采用流式细胞术鉴定干细胞；用复方扶芳藤合剂干预，基因芯片及抗体芯片检测干预前后造血龛内干细胞的基因表达谱及分泌的细胞因子，全面分析差异显著基因及细胞因子的变化，探讨复方扶芳藤合剂调控造血龛造血的分子作用机制，差异显著基因和细胞因子用qPCR和ELISA验证；对差异显著基因进行表达调控网络分析，了解复方扶芳藤合剂整体调控造血龛造血的信号途径，揭示复方扶芳藤合剂益气生血中医理论的科学内涵，为复方中药配合干细胞共移植治疗疑难血液病提供理论支持。

复方扶芳藤合剂具有益气健脾，补血养心的功效。国内外研究发现造血微环境，造血龛（Niche）多由间充质干细胞（MSCs）及造血干细胞（HSC）构成，Niche内MSCs和HSC通过细胞接触产生作用。前期我们研究发现复方扶芳藤合剂对小鼠的外周血HSC及大鼠骨髓的MSCs有促增殖及调控作用，我们推测其对由MSCs+HSC构成的Niche同样会产生影响。为了证实这一假说,我们首先建立了稳定的SD大鼠骨髓间充质干细胞（BMSCs）和造血干细胞（HSC）分离及共培养方法，筛选出最佳体外模拟大鼠“造血龛”的共培养模式。为进一步研究复方扶芳藤合剂对“造血龛”的作用机制，我们用基因芯片检测“龛”内MSCs与HSC被复方扶芳藤合剂含药血清干预后的基因组表达谱，得到的表达基因数量分别为12093与13620个，其中“龛”内MSCs的差异最显著上调基因有31个，下调的有29个，信号通路分析其主要富集在与调控干细胞增殖分化等信号通路。GO分析发现，在复方扶芳藤合剂干预后“龛”内的MSCs的与外泌体相关的mRNA富集非常显著（FDR<0.01）。差异特显著上调基因进行qPCR验证，验证结果显示9个基因上调明显，结果与芯片吻合；我们用蛋白芯片检测发现“龛”内上清液里的22个与造血相关的细胞因子发生变化，其中上调15个，下调7个，上调最显著的是IL-3 、IL-6、GM-CSF 及VEGF等。实验证明，复方扶芳藤合剂大鼠含药血清对体外模拟“造血龛”中MSCs的增殖具有调控作用，能增加MSCs的数量，通过增加MSCs的数量促进HSC的增殖，这种促增殖调控作用是通过多靶点、多信号途径实现的。MSCs和HSC在中医属于“先天之精”和“后天之精”范畴，其对MSCs和HSC的促增殖调控作用，为中医理论揭示了“脾为后天之本，为气血生化之源”的科学内涵，为临床利用复方中药配合HSC+MSCs共移植治疗疑难血液病提供新思路。