Notch信号通路调控骨髓间充质干细胞向肝样细胞分化机制研究

Bone marrow mesenchymal stem cells(BMSCs) have the advantages of multi-line directional differentiation capacity and low immunogenicity, has been widely used in regenerative medicine.In the message of large number of basic and clinical research,it suggest that BMSCs can be induced to differentiate into functional hepatocyte-like cells in vitro and in vivo,but we know less about its differentiation mechanism.Notch signaling pathway plays a key role in the regulation of the BMSCs directed differentiation process, but it reported less about the mechanism involved in the process of directed differentiation to hepatocyte-like cells.Therefore,in this study we propose to make sure the expression difference of some key factors(Notch1、Jagged1、PS1、NICD、RBP-jk、Hes1)in classic Notch signaling pathway in BMSCs directed differentiation,on the basis of preliminary work,to make sure these factors whether involved in Notch signaling pathway.To investigate the mechanism of Notch signaling pathway in BMSCs induced differernted to hepatocyte-like cells,we use over expression and RNAi techology on different site to active or block signal conduct,and observe the effect of over expression and RNAi to hepatocyte-like cells differentiation，in order to get new method for the clinical to guidance us directed differentiation of BMSCs,to provide a theoretical basis for a variety of end-stage liver disease treatment, and also for new drug targets to provide an initial basis for the work.

骨髓间充质干细胞（BMSCs）具有多系定向分化能力和低免疫原性等优点，在再生医学中得到广泛应用。大量实验研究及申请者的前期工作表明，BMSCs在体内外均可诱导分化为有功能的肝样细胞，但对其分化机制研究较少。Notch信号通路在调控BMSCs定向分化过程中起关键作用，但尚无涉及向肝样细胞定向分化机制的报道。故本研究在前期工作基础上拟检测经典Notch信号通路中关键信号分子Notch1、Jagged1、PS1、NICD、RBP-jk、Hes1在体外诱导BMSCs向肝样细胞分化过程中表达变化，确定参与分化的Notch信号分子。利用体外基因过表达和基因沉默方法分别从不同位点激活或阻断其信号传导，观察其对肝样细胞的分化影响，阐明Notch信号通路调控BMSCs向肝样细胞定向分化作用机制，为临床获得BMSCs定向分化提供新的方法指导，为各种终末期肝病治疗提供理论基础，也为新药靶标发现提供前期工作基础。

细胞移植治疗已成为急慢性肝病的有效治疗手段，BM-MSCs来源的肝样细胞有望成为细胞移植治疗肝病的种子细胞，本项研究也成为该领域的热点。我们通过合理的实验设计，摸索出了将BM-MSCs成功有效的诱导为肝样细胞的三步法诱导方案，并通过PCR技术、免疫荧光细胞化学染色和Western Blot等方法在基因及蛋白水平充分证明了诱导所得细胞的肝细胞特点。与此同时，我们发现在BM-MSCs向肝样细胞分化的过程中，Notch1的表达有显著的降低，提示Notch信号通路的活动可能对BM-MSCs向肝样细胞分化有抑制作用。我们设计应用RNA干扰技术，干扰Notch1的表达，重复上述诱导分化过程，进一步明确Notch通路在BM-MSCs向肝样细胞分化的过程中的作用。我们已经合成拥有携带有Notch1 shRNA的质粒，成功摸索出了该质粒的合理用量及其与转染试剂用量的比例关系。并且筛选出了能有效干扰Notch1基因表达的质粒，为下一步的实验奠定了坚实的基础。目前，国内外关于间充质干细胞向肝样细胞分化相关机制的研究很少，我们的研究成果将弥补这一空缺，为下一步的临床应用提供有意义的基础。