Notch信号通路调控当归补血汤协同肌卫星细胞移植促进造血重建的机理研究

Prophase study proved that autologous source muscle satellite cells (MSC) with the multiplex potential differentiation capability could differentiate into hematopoietic cells under the induction of hematopoietic microenvironment. Our previous study which subsidized by NSFC indicated that Danggui Buxue Decoction (DBD) has the capability to improve the body's hematopoietic microenvironment effectively and affect numberous factors of hematopoietic process. In order to provide the experiment basis of screening seed stem cell for clinical application, through in vivo and vitro experiments, by using molecular biology technique, like fluorescent quantitation, Real time PCR, Western blot, etc,we are going to study the gene expression changes of those key genes in the Notch signal pathway, such as Notch1,Jagged1, and its downstream gene:CyclinD1,bcl-2 and bax， together with their sub-cellular localization of these proteins. In this project, we expect to find role targets and reveal the molecular regulation mechanism of Notch signal pathway that can control MSC to differentiate into hematopoietic cells under the inducing of hematopoietic microenvironmen by using molecular biotechnology that can detect critical gene expression of Notch signal pathway. Thus we could find the certain pathway of Notch signal which could accurately controll the process of MSCs differentiation into hematopoietic cells on the gene level.

研究表明：具多向分化潜能的肌卫星细胞（MSC）在特定微环境中可分化为造血细胞。通过前基金项目研究结果提示：当归补血汤有明显的改善造血微环境的能力，从多环节影响造血过程,有可能通过影响Notch通路各环节干预MSC向造血细胞的增殖分化过程。此次拟通过在体和离体实验,运用免疫荧光、real-time PCR、Westen Blot等生物学技术方法，基于Notch信号途径，检测Notch通路中关键基因Notch1、Jagged1、HES1及其调控的靶基因PCNA、Pax7、MyoD、CD45、c-kit及Sca-1的表达及蛋白的亚细胞定位，阐明MSC在造血诱导微环境中，Notch信号系统对MSC定向诱导分化为造血细胞的分子调控机理及调控通路的作用靶点；找到调节Notch信号途径的方式，从而可通过改变Notch信号，精确调控MSC向造血细胞分化的过程和比例，为种子细胞的筛选和临床应用提供实验依据

Notch信号通路对骨髓内的造血细胞具有调控作用，当归补血汤有明显的改善造血微环境的能力，能够协同Notch信号通路调控肌源性干细胞的多能分化。. 实验分为在体动物实验和离体细胞实验，采用形态学、免疫组化、细胞分子生物学的检测方法，探讨Notch 信号在体外受体鼠造血重建过程中及造血诱导微环境中的表达情况，明晰notch对MDSCs 定向诱导分化为造血细胞的分子调控机理及调控通路的作用靶点；检测 Notch 通路中关键基因 Notch1、Jagged1、HES1 、Notch4、Delata4及其调控的靶基因 PCNA、Pax7、MyoD、CD45、c-kit 及 Sca-1 的表达及蛋白的亚细胞定位，寻找调节 Notch 信号途径的方式，从而可通过改变Notch 信号，精确调控 MSC 向造血细胞分化的过程和比例，为种子细胞的筛选和临床应用提供实验依据。. 结果显示：Notch1、Jagged1、Hes1对骨髓内的造血细胞具有调控作用，Notch4、Delta4在骨髓造血干细胞移植和肌源性干细胞移植中的作用不同。. 结论：经当归补血汤干预后，移植MDSCs的受体鼠造血功能更易于恢复重建，notch信号通路相关基因表现出不同程度的调控作用；当归补血汤与notch信号协同能够促进MDSCs的造血分化。