摘要
目的 探讨以外泌体为载体将miR-124递送给神经元的可能性及其对轴突再生相关因子表达的影响. 方法 将装载miR-124的质粒转染至HEK293细胞,并应用实时定量PCR（qPCR）鉴定转染效果.从转染了miR-124的HEK293细胞培养液上清液提取外泌体,应用电镜观察、Western blotting及qPCR鉴定外泌体及其中是否内含miR-124.将C57BL/6孕鼠胚胎皮层神经元体外培养7d后分为4组：对照组、损伤组、损伤＋不含miR-124外泌体组、损伤＋内含miR-124外泌体组,其中后3组用移液器塑料滴头在培养板内划割造成机械性损伤,后2组分别将不含或内含miR-124的外泌体加入培养板内,继续培养72 h后分别应用qPCR和Western blotting检测各组神经元中miR-124及神经纤毛蛋白-1（NRP-1）、微管相关蛋白Tau、生长相关蛋白43（GAP-43）蛋白的表达. 结果 成功获得了内含miR-124的外泌体.损伤＋内含miR-124外泌体组神经元中miR-124及NRP-1、GAP-43蛋白表达量均明显高于其他3组,损伤组和损伤＋不含miR-124外泌体组神经元中miR-124及NRP-1、GAP-43蛋白表达量均明显高于对照组,差异均有统计学意义（P＜0.05）. 结论 外泌体可递送miR-124至机械性损伤后的小鼠皮层神经元并提高神经元中轴突再生相关因子NRP-1、GAP-43的表达.
Objective To explore the influence of exosome-derived miR-124 on the molecular expression related to axonal regeneration after mechanical damage to cortical neurons in mice,aiming to provide experimental data for intervention in neurogenesis after traumatic brain injury （TBI）.Methods The plasmid loaded with miR-124 was used to transfect the HEK293 cell line.The transfection effect was identified by real time Polymerase Chain Reaction （qPCR）.The exosomes were isolated from the supematant of cultured transfected HEK293 cell line by the SBI isolation kit.The isolated exosomes were identified by electron microscopy and Western blotting,and the involved miR-124 in the exosomes was identified by qPCR.After the cortical neurons were isolated from the pregnant mice （14-17-day old） and cultured for 7 days,they were divided into 4 groups：control,damage,damage ＋ exosomes without miR-124 and damage ＋ exosomes with miR-124.The Petri dishes were manually scratched with a 10 μL plastic stylet needle to construct a mechanical damage in vitro in the latter 3 groups.The isolated exosomes without or with miR-124 were added into the cultured medium for culture for 72 h in the latter 2 groups,respectively.The expression ofmiR-124,NRP-1,Tau and Gap-43 was measured by qPCR and Western blotting respectively.Results The exosomes containing miR-124 were successfully obtained by plasmid transfection and the SBI isolation kit.The expression levels of miR-124,NRP-1 and Gap-43 in the damage ＋ exosomes with miR-124 group were elevated significantly greater than in the other 3 groups （P＜0.05）.The expression levels ofmiR-124,NRP-1 and Gap-43 in the damage group and damage ＋ exosomes without miR-124 group were elevated significantly greater than in control group （P＜0.05）.Conclusions The exosomes may transmit miR-124 to the cortical neurons in mice after mechanical damage and increase the expression ofmiR-124,NRP-1 and Gap-43 in the cortical neurons in mice.
作者
杨永祥
叶玉勤
苏鑫洪
张欣
孔垂广
白威
贺晓生
Yang Yongxiang;Ye Yuqin;Su Xinhong;Zhang Xin;Kong Chuiguang;Bai Wei;He Xiaosheng(Department of Neurosurgery, Xijing Hospital, A irforce Military Medical University, Xi＇an 710032, China)
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2018年第5期440-444,共5页
Chinese Journal of Neuromedicine
基金
国家自然科学基金（81471264）
