hUC-MSCs来源外泌体联合肾脑复元汤激活Wnt3a/β-catenin通路减轻NSCs损伤的研究  

Study on hUC-MSCs Derived Exosomes Combined with Shennao Fuyuan Decoction Reduced NSCs Injury through Activation of Wnt3a/β-catenin Pathway

作　　者：李旭敏 商燕 谢丽华 胡国恒[2] LI Xumin;SHANG Yan;XIE Lihua;HU Guoheng(Hunan University of Chinese Medicine,Changsha 410208,China;The First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,China)

机构地区：[1]湖南中医药大学,湖南长沙410208 [2]湖南中医药大学第一附属医院,湖南长沙410007

出　　处：《中医药信息》2023年第5期12-20,共9页Information on Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81573941);湖南省自然科学基金面上项目(2020JJ4476);湖南中医药大学校级科研基金项目(2018XJJJ29);湖南中医药大学中医学国内一流建设学科项目(2018ZXY03);胡国恒名医传承工作室项目(湘中医药函[2018]37号);国家中医药管理局“王行宽全国名中医传承工作室”建设项目(国中医药办人教函[2018]119号)。

摘　　要：目的:探讨人脐带间充质干细胞(hUC-MSCs)来源的外泌体联合肾脑复元汤减轻氧葡萄糖剥夺/复氧(OGD/R)诱导的神经干细胞(NSCs)损伤的作用机制。方法:组织块法分离培养SD乳鼠的NSCs;免疫荧光鉴定NSCs标记物Nestin的表达;流式细胞术鉴定hUC-MSCs,分离外泌体,透射电镜观察;采用OGD/R构建NSCs损伤模型;CCK8法、EdU法和流式细胞术用于评估NSCs的增殖和凋亡;qRT-PCR、蛋白质印迹法和免疫细胞化学法分析Wnt3a/β-catenin通路相关基因的表达。结果:本研究成功分离得到并鉴定了NSCs和hUC-MSCs,以及hUC-MSCs来源的外泌体。OGD/R诱导NSCs细胞活力和增殖率下降,促进细胞凋亡,抑制Wnt3a、β-catenin、Cyclin D1和TCF4基因和蛋白表达。与OGD/R模型组比较,肾脑复元汤、外泌体,以及二者联用显著增加NSCs的细胞活力和增殖率,抑制凋亡,促进Cyclin D1、β-catenin、Wnt3a和TCF4基因和蛋白的表达。结论:hUC-MSCs来源外泌体联合肾脑复元汤通过促进NSCs细胞增殖减轻OGD/R诱导的NSCs细胞损伤,可能与Wnt3a/β-catenin通路活化有关。Objective:To investigate the mechanism of human umbilical cord mesenchymal stem cells(hUCMSCs)derived-exosomes combined with Shennao Fuyuan Decoction(SFD)in alleviating oxygen-glucose deprivation/reoxygenation(OGD/R)induced neural stem cells(NSCs)injury.Methods:The NSCs of SD suckling rats were isolated and cultured by tissue block method.The expression of NSCs marker Nestin was determined by immunofluorescence.hUC-MSCs were identified by flow cytometry.Exosomes were isolated and observed by transmission electron microscope.The damage model of NSCs was established by OGD/R.Proliferation and apoptosis of NSCs were assessed by CCK8,EdU and flow cytometry.The expressions of Wnt3a/β-catenin pathway related genes were analyzed by quantitative real-time PCR(qRT-PCR)technique,Western blot method and immunohistochemistry method.Results:NSCs,hUC-MSCs and hUC-MSCs-derived exosomes were successfully isolated and identified.OGD/R induced the decreased cell activity and proliferation rate of NSCs,promoted the apoptosis,and inhibited the gene and protein expressions of Wnt3a,β-catenin,Cyclin D1 and TCF4.Compared with those in the OGD/R model group,the cell viability and proliferation rate of NSCs were significantly increased,the apoptosis was inhibited,and the gene and protein expressions of Cyclin D1,β-catenin,Wnt3a and TCF4 were promoted by the interventions of SFD,exosomes,and the combination of SFD and exosomes.Conclusion:hUC-MSCs-derived exosomes combined with SFD could reduce OGD/R induced cell damage of NSCs by promoting NSCs proliferation,which may be related to the activation of Wnt3a/β-catenin pathway.

关 键 词：肾脑复元汤 人脐带间充质干细胞(hUC-MSCs) 外泌体 氧葡萄糖剥夺/复氧(OGD/R) WNT3A β-catenin 

分 类 号：R285.5[医药卫生—中药学]