葛根素促进小鼠胚胎干细胞源工作心肌细胞的分化及其调控机制-生理学专业论文.docxVIP

华中科技大学博士学位论文 华 中 科 技 大 学 博 士 学 位 论 文 6 6 Inducible effect of puerarin on embryonic stem cells differentiation and regulatory mechanism Abstract Aims: Currently CMs derived from stem (ES) cells represent the most promising CMs for cardiac regenerative medicine.It is important to screen and identify chemical compounds that induce high efficiency cardiac differentiation. Radix puerariae is a traditional Chinese herbal medicine to treat many cardiac diseases. The aim of this study was to investigate the possible inducible effects of puerarin on embryonic stem cells differentiation and regulatory mechanism in vitro. Methods: Effect of puerarin on proliferation of murine ES cells were analyzed via MTT assay. then were differentiated using The classic hanging drop - Suspension - adherent three step differentiation protocol with or without 1μ mol/L、10μ mol/L、100μ mol/L puerarin. Differentiating ES cells were analyzed via morphological analysis for diameter of embryonic bodys (EBs) and percentage of beating EBs, immunofluorescence staining for a- sarcomeric -actinin, RT-PCR for specific genes of different mesodern, western blot for serine-threonine kinase (Akt) and phosphorylation of the serine-threonine kinase (P-Akt). Results: Puerarin （100μ mol/L）resulted in significantly increased number of cardiac cells and percentage of beating EBs,100μ mol / L puerarin was the optimal concentration . Puerarin resulted in a significantly increased the number of a- sarcomeric –actinin positive cells on differentiation day 12. RT-PCR results showed expressions of endoderm, cardiac (mesoderm) and endothelial cells specific gene and decreased expressions of neuronal cells (ectoderm) specific gene at early differentiation stages. Furthermore, we confirmed that embryoid body (EB)-size increased by puerarin might affect the direction of differentiation. Puerarin also facilitated phosphorylation of the serine-threonine kinase (Akt). 31 31 Puerarin increases cardiac differentiation of murine