益肾Ⅰ号靶向Notch信号促帕金森病NSCs向多巴胺神经元分化

Directed differentiation of neural stem cells（NSCs）into dopaminergic neurons for use in cell therapy of Parkinson's disease (PD) has been a research focus.Under the instruction of"the kidney dominating bone and nourishing marrow" theory, we have demonstrated Yishen Ⅰfound in P.testudinis can induce differentiation of NSCs into dopaminergic neurons and descreased the expression of Notch1. So we set out to explore the regulatory mechanism that Yishen I induces differentiation of NSCs into dopaminergic neurons through Notch signaling pathway. First, to investigate which compound of Yishen Ⅰ has the activing effect on Notch signaling, NSCs were transfected with Hes1 promoter reporter construct , Notch downstream target gene, and then either left unstimulated or stimulated with Yishen Ⅰ，A dual lueiferase reporter system was applied to detect transcriptional activity of Hes1; Second, to assess Whether Notch signaling involves effect of the compound of Yishen Ⅰ on differentiation of NSCs into dopaminergic neurons. Using RNAi or the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S- phenylglycine t-butyl ester (DAPT) to interfere with Notch activity, Western blot and RT-PCR were used to detect Notch signaling Pathway molecular，PS1,RBP-Jk and Hes1 expression; Third, to study the correlation of effects of Yishen Ⅰ on the differentiation of endogenous neural stem and the expression of Notch in rats models of Parkinson's disease in vivo. We used bromodeoxyuriding(BrdU)/Nestin to label the endogenous NSCs . Immunohistochemical stain show that TH and Notch staining .Studying relationship between Yishen Ⅰ and Notch signaling may provide new strategy to bushen in therapy of Parkinson's disease.

如何促神经干细胞定向分化为多巴胺神经元,是帕金森病治疗的关键问题。依据中医"肾主骨生髓"理论及前期工作基础，益肾Ⅰ号可促进神经干细胞定向分化为多巴胺神经元细胞，抑制Notch1分子的表达。因此, 提出"益肾Ⅰ号有效成分群选择性抑制Notch信号通路所产生的整合作用，促进神经干细胞逃逸旁侧抑制而定向分化为多巴胺神经元细胞"。①阐明Notch下游靶基因Hes1反应元件，确定益肾Ⅰ号何种成分激活Notch信号通路;②阐明Notch何种信号组分介导,观察益肾Ⅰ号是否依赖Notch信号通路起作用;③阐明益肾Ⅰ号对PD模型大鼠脑组织中内源性NSCs向多巴胺神经元分化情况与Notch信号的相关性,为中医补肾法在帕金森病治疗中提供新思路。

项目背景：如何促神经干细胞定向分化为多巴胺神经元，是帕金森病治疗的关键问题。根据中医“肾主骨生髓”理论及前期工作基础，益肾I号可促进神经干细胞定向分化为多巴胺神经元细胞，抑制Notch1分子的表达。因此，提出“益肾I号有效成分群选择性抑制Notch信号通路缩产生的整合作用，促进神经干细胞逃逸旁侧抑制而定向分化为多巴胺神经元细胞”。.主要研究内容：①阐明Notch下游靶基因Hes1反应元件，确定益肾I号何种成分激活Notch 信号通路；②阐明Notch何种信号组分介导，观察益肾I号是否依赖Notch信号通路起作用；③阐明益肾I号对PD模型大鼠脑组织中内源性NSCs向多巴胺神经元分化情况与Notch信号的相关性。.重要结果：本研究证明益肾I号的主要成分——龟板有效成分PTE（2B）、龟板提取物十八酸乙酯（S6）、甾酮（S9），能够促进外源性和内源性神经干细胞向多巴胺神经元分化，抑制Notch1及Notch下游靶基因Hes1的表达。.关键数据：发表SCI论文1篇，中文期刊论文2篇。.科学意义：本研究探讨Notch信号通路在益肾I号促神经干细胞向多巴胺神经元定向分化中的调控机制，阐明益肾I号对PD模型大鼠内源性神经干细胞向多巴胺神经元分化情况与Notch信号的相关性，为中医补肾法在帕金森病治疗中提供新思路。