毛蕊异黄酮对人诱导多能干细胞内皮分化的影响及机制  

Effects and mechanisms of calycosin on endothelial differentiation of human induced pluripotent stem cells

作　　者：崔胜男 刘传国 杨雯晴[1,2] 郑志娟[2] 张丹[2,3] Cui Shengnan;Liu Chuanguo;Yang Wenqing;Zheng Zhijuan;Zhang Dan(Innovative Institute of Traditional Chinese Medicine;Experimental Center,Shandong University of Traditional Chinese Medicine,Jinan 250355,Shandong Province,China;Key Laboratory of Traditional Chinese Medicine Classical Theory,Ministry of Education,Shandong University of Traditional Chinese Medicine,Jinan 250355,Shandong Province,China)

机构地区：[1]山东中医药大学中医药创新研究院,山东省济南市250355 [2]山东中医药大学实验中心,山东省济南市250355 [3]中医药经典理论教育部重点实验室,山东省济南市250355

出　　处：《中国组织工程研究》2024年第19期3031-3036,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金青年科学基金资助项目(82174337),项目负责人:杨雯晴;山东“高校20条”资助项目(2020GXRC017),项目参与人:崔胜男,郑志娟,张丹。

摘　　要：背景:内皮损伤是心血管疾病的诱因之一,人诱导多能干细胞易于获得、分化能力强、排异性较小,其内皮分化细胞可被用作心血管疾病研究的理想细胞。目的:探讨毛蕊异黄酮对人诱导多能干细胞定向内皮分化的作用及机制,为微血管再生提供技术支持。方法:将人诱导多能干细胞分为对照组与毛蕊异黄酮1.25,2.5μg/mL组,进行内皮定向诱导分化。诱导分化8 d后,采用流式细胞术检测各组细胞内皮细胞标志物CD144阳性率,免疫荧光技术检测CD144、CD31荧光表达。利用慢病毒RNAi-GFP puromycin沉默人诱导多能干细胞Piezo1 mRNA,再进行内皮定向诱导分化,诱导分化8 d后,采用流式细胞术检测分化细胞CD144阳性率,qPCR检测CD144、Piezo1、MEK的mRNA表达水平。结果与结论:①与对照组相比,毛蕊异黄酮1.25,2.5μg/mL组CD144阳性率显著升高(P<0.05);毛蕊异黄酮2.5μg/mL组CD144、Piezo1、MEK mRNA表达水平提高(P<0.05);毛蕊异黄酮2.5μg/mL组CD144(P<0.01)和CD31(P<0.001)荧光表达显著升高;②与shNT组相比,shNT+毛蕊异黄酮1.25,2.5μg/mL组CD144阳性率和CD144、Piezo1、MEK mRNA表达显著升高(P<0.05),与shPiezo1组相比,shPiezo1+毛蕊异黄酮1.25,2.5μg/mL组CD144阳性率和CD144、Piezo1、MEK mRNA表达无显著变化(P>0.05);③实验结果提示2.5μg/mL毛蕊异黄酮能够促进人诱导多能干细胞内皮分化,毛蕊异黄酮可以通过靶向调节Piezo1表达水平,促进下游MEK表达,从而促进人诱导多能干细胞内皮分化。BACKGROUND:Endothelial injury is one of the causes of cardiovascular diseases.Human induced pluripotent stem cells are easy to obtain,have strong differentiation ability,and have less exclusiveness,and their endothelial differentiated cells can be used as ideal cells for cardiovascular disease research.OBJECTIVE:To investigate the effect and mechanism of calycosin on endothelial differentiation of human induced pluripotent stem cells and to provide technical support for microvascular regeneration.METHODS:Human induced pluripotent stem cells were divided into control group and calycosin group(1.25,2.5μg/mL),and growth factors were added to induce single-layer endothelial differentiation.After the induction of differentiation for 8 days,the positive rate of endothelial cell marker CD144 was detected by flow cytometry.Fluorescent expressions of CD144 and CD31 were detected by the immunofluorescence method.Lentivirus RNAi GFP puromycin was used to silence human-induced pluripotent stem cell Piezo1 mRNA followed by endothelial directed differentiation.After 8 days of differentiation,the positive rate of CD144 in differentiated cells was detected by flow cytometry.The mRNA expression levels of CD144,Piezo1 and MEK were detected by qPCR.RESULTS AND CONCLUSION:(1)Compared with the control group,the positive rate of CD144 was significantly increased in the 1.25 and 2.5μg/mL calycosin groups(P<0.05).The expressions of CD144,Piezo1,and MEK mRNA were increased in the 2.5μg/mL calycosin group(P<0.05).The fluorescence expressions of CD144(P<0.01)and CD31(P<0.001)were significantly increased in the 2.5μg/mL calycosin group.(2)Compared with the shNT group,CD144 positive rate and CD144,Piezo1,MEK mRNA expressions were significantly increased in the shNT+calycosin 1.25,2.5μg/mL groups(P<0.05).Compared with the shPiezo1 group,the positive rate of CD144 and mRNA expressions of CD144,Piezo1 and MEK had no significant changes in the shPiezo1+calycosin 1.25,2.5μg/mL groups(P>0.05).(3)It is concluded that 2.5μg/mL calycosin promo

关 键 词：毛蕊异黄酮 人诱导多能干细胞 内皮细胞 内皮分化 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学] R542.2