益肾化浊方条件培养基对神经干细胞增殖分化影响的研究

Effect of Yishen Huazhuo conditioned medium regulating NSCs proliferation and differentiation by JAK / STAT signaling pathway

目的从JAK/STAT信号转导通路,探讨益肾化浊通过微环境调控神经干细胞(NSCs)增殖与分化,为益肾化浊方治疗AD提供部分实验依据。方法分离提取孕14 d昆明小鼠胚胎NSCs,以5μmol·L-1β淀粉样蛋白25-35(Aβ25-35)干预建立AD细胞模型,并以不同浓度益肾化浊方共培养48h后,分别收集各组细胞培养基,使用基础培养基稀释成10%浓度条件培养基,培养正常神经干细胞,分为空白对照组,正常条培组,Aβ条培组,复方0.1μg·ml-1条培组,复方1μg·ml-1条培组,复方10μg·ml-1条培组,共6组。采用CCK-8法检测益肾化浊方条件培养基对NSCs增殖与细胞活性的影响,免疫荧光法与Western Blot法检测其对NSCs分化的影响;Western Blot与PCR法检测JAK/STAT信号转导通路关键靶点蛋白与基因表达的变化。结果与Aβ条培组相比,益肾化浊方条件培养基可促进NSCs增殖,提高其细胞活性;同时可促进NSCs向神经元分化,抑制其向星形胶质细胞分化,差异具有统计学意义(P<0.05)。复方0.1μg·ml-1条培组均可明显降低STAT3、P-STAT3、Smad1、GFAP蛋白表达,下调GFAP、STAT3、Smad1基因同时上调ngn1基因表达,促进NSCs向神经元分化,差异具有统计学意义(P<0.05)。结论益肾化浊方条件培养基可通过调控JAK/STAT信号转导通路,提高NSCs活性,促进神经元分化,延缓AD进展。

Objective To discuss the effects of Yishen Huazhuo decoction regulating neural stem cells（ NSCs） proliferation and differentiation through microenvironment by JAK / STAT signaling pathway,and provide some experimental basis for Yishen Huazhuo Decoction in the treatment of AD. Methods Extracting NSCs from the embryos of 14 days of Kunming mice pregnancy,to establish cell model of Alzheimer＇s disease（ AD） by Aβ25- 35,NSCs were cultured with different levels of Yishen Huazhuo Decoction for 48 hours,to collect cell culture medium respectively. Diluted to 10% conditioned medium with basic medium,to cultivate normal NSCs,divided into control group,conditioned medium group,Aβ25- 35 conditioned medium group,0. 1 μg·ml- 1YSHZ group,1 μg·ml- 1YSHZ group,1 0μg·ml- 1YSHZ group. The effect of Yishen Huazhuo Decoction on proliferation of NSCs was observed by CCK- 8 method,and its differentiation were determined by immune fluorescence and Western Blot method. Then,Western Blot and RT- PCR methods were used to observe the effect of key target protein and gene expression of JAK / STAT signaling pathway. Results Compared with Aβ25- 35 conditioned medium group,YSHZ conditioned medium groups could increase NSCs proliferation,promote the differentiation of neurons while suppressing astrocyte（ P〈0. 05）,which was statistically different significant. GFAP,STAT3,P- STAT3,Smad1 protein and gene expression of 0. 1 μg · ml- 1YSHZ group were statistically redused,while ngn1 gene expression was up- regulated（ P〈0. 05）. Conclusion YSHZ conditioned medium could improve the activity of NSCs,and promote the differentiation of neurons by regulating key target of JAK / STAT signaling pathway,in order to suspend AD progress.