【作者】 张妙齐； 王丙云； 李东升； 阮慧敏； 叶彩玲； 李星颖； 何诗； 陈胜锋； 白银山； 陈志胜； 刘璨颖； 计慧琴；

【Author】 Zhang Miaoqi;Wang Bingyun;Li Dongsheng;Ruan Huimin;Ye Cailing;Li Xingying;He Shi;Chen Shengfeng;Bai Yinshan;Chen Zhisheng;Liu Canying;Ji Huiqin;School of Life Science and Engineering,Foshan University;VetCell Biotechnology Co.,LTD;

【机构】 佛山科学技术学院生命科学与工程学院； 维赛(佛山)生物科技有限公司；

【摘要】 为研究犬脐带间充质干细胞外泌体(Umbilical cord mesenchymal stem cell exosomes,UC-MSC-Exo)中携带的cfa-miR-21促进犬血管内皮细胞(Vein endothelial cells,VECs)增殖及迁移的作用机制,本实验以犬VECs为模型,通过qRT-PCR筛选出cfa-miR-21最佳转染方案,然后使用CCK-8法、EdU法和划痕实验,检测cfa-miR-21对细胞的增殖及迁移的调控作用;通过生物信息学分析软件预测靶基因,并利用qRT-PCR、双荧光素酶报告基因系统验证目的基因,再通过Western Blot检测下游MAPK/ERK信号通路蛋白的表达情况,最后通过敲减靶基因,检测细胞增殖及迁移相关基因的表达。实验结果表明,cfa-miR-21可促进犬血管内皮细胞的增殖和迁移,其可通过靶向作用于SPRY1,激活MAPK/ERK信号通路从而发挥生物功能。在此过程中,VEGFA、CCND1、PCNA和N-Cadherin的上调也参与了血管内皮细胞功能的调控。更多还原

【Abstract】 In order to study the mechanism of cfa-miR-21 carried by canine umbilical cord mesenchymal stem cell exosomes(UC-MSC-Exo) in promoting the proliferation and migration of canine vascular endothelial cells(VECs),the following studies were carried out in this experiment.In this study,canine VECs was used as a model,the best transfection scheme of cfa-miR-21 transfection reagent was screened by qRT-PCR,and the regulatory effect of cfa-miR-21 on cell proliferation and migration was detected by cell counting kit-8(CCK-8),EdU cell proliferation imaging and scratch test.The target gene was predicted by bioinformatics analysis software,and the target gene was verified by qRT-PCR and dual luciferase reporter gene system.Then the expression of downstream MAPK/ERK signal pathway protein was detected by Western Blot.Finally,the expression of cell proliferation and migration related genes was detected by knocking down the target gene.The results suggest that cfa-miR-21 can promote the proliferation and migration of canine vascular endothelial cells,and cfa-miR-21 can exert its biological function by targeting SPRY1 and activating MAPK/ERK signal pathway.In this process,the up-regulation of VEGFA,CCND1,PCNA and N-Cadherin are also involved in the regulation of vascular endothelial cell function.更多还原