miR-27a过表达的血管内皮细胞来源外泌体改善股骨头坏死实验研究  

Experimental study on improvement of osteonecrosis of femoral head with exosomes derived from miR-27a-overexpressing vascular endothelial cells

作　　者：张根生 刘瑞宇[2] 党晓谦[2] 刘继超 焦海斌 ZHANG Gensheng;LIU Ruiyu;DANG Xiaoqian;LIU Jichao;JIAO Haibin(Department of Orthopaedics,3201 Hospital of Xi'an Jiaotong University Health Science Center,Hanzhong Shaanxi,723000,P.R.China;Department of Orthopaedics,the Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an Shaanxi,710004,P.R.China)

机构地区：[1]西安交通大学医学部附属三二〇一医院骨科,陕西汉中723000 [2]西安交通大学第二附属医院骨科,西安710004

出　　处：《中国修复重建外科杂志》2021年第3期356-365,共10页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：陕西省重点研发项目(2019SF-168)。

摘　　要：目的探究miR-27a过表达的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)来源外泌体exo(miR-27a)能否促进骨再生并改善糖皮质激素(glucocorticoids,GC)诱导的股骨头坏死(osteonecrosis of femoral head,ONFH)(GC-ONFH)。方法构建exo(miR-27a),并通过透射电镜、纳米颗粒跟踪分析、Western blot和实时荧光定量PCR(real-time fluorescent quantitative PCR,qRT-PCR)进行鉴定。采用qRTPCR评估exo(miR-27a)递送miR-27a至成骨细胞MC3T3-E1的效果,ALP染色、茜素红染色及qRT-PCR评估其对MC3T3-E1细胞成骨的影响。行双荧光素酶报告基因检测验证miR-27a靶向Dickkopf WNT信号通路抑制剂2(Dickkopf WNT signaling pathway inhibitor 2,DKK2)是否为潜在机制,并在MC3T3-E1细胞内通过qRT-PCR、Western blot及茜素红染色进一步验证该机制。最后,通过SD大鼠的GC-ONFH模型验证exo(miR-27a)对ONFH的保护作用。结果透射电镜、纳米颗粒跟踪分析、Western blot和qRT-PCR检测示exo(miR-27a)构建成功。exo(miR-27a)可将miR-27a有效递送至MC3T3-E1细胞并提高其成骨能力。双荧光素酶报告基因检测示miR-27a通过直接靶向DDK2促进成骨。动物实验Micro-CT及HE染色结果显示,尾静脉注射exo(miR-27a)改善了SD大鼠GC-ONFH模型的骨坏死情况。结论exo(miR-27a)可以促进骨再生,并在一定程度上改善GCONFH。Objective To investigate whether exosomes derived from miR-27 a-overexpressing human umbilical vein endothelial cells(HUVECs)—exo(miR-27 a)can promote bone regeneration and improve glucocorticoids(GC)induced osteonecrosis of femoral head(ONFH)(GC-ONFH).Methods The exo(miR-27 a)were intended to be constructed and identified by transmission electron microscopy,nanoparticle tracking analysis,Western blot,and realtime fluorescent quantitative PCR(qRT-PCR).qRT-PCR was used to evaluate the effect of exo(miR-27 a)in delivering miR-27 a to osteoblasts(MC3 T3-E1 cells).Alkaline phosphatase staining,alizarin red staining,and qRT-PCR were used to evaluate its effect on MC3 T3-E1 cells osteogenesis.Dual-luciferase reporter(DLRTM)assay was used to verify whether miR-27 a targeting Dickkopf WNT signaling pathway inhibitor 2(DKK2)was a potential mechanism,and the mechanism was further verified by qRT-PCR,Western blot,and alizarin red staining in MC3 T3-E1 cells.Finally,the protective effect of exo(miR-27 a)on ONFH was verified by the GC-ONFH model in Sprague Dawley(SD)rats.Results Transmission electron microscopy,nanoparticle tracking analysis,Western blot,and qRT-PCR detection showed that exo(miR-27 a)was successfully constructed.exo(miR-27 a)could effectively deliver miR-27 a to MC3 T3-E1 cells and enhance their osteogenic capacity.The detection of DLRTM showed that miR-27 a promoted bone formation by directly targeting DDK2.Micro-CT and HE staining results of animal experiments showed that tail vein injection of exo(miR-27 a)improved the osteonecrosis of SD rat GC-ONFH model.Conclusion exo(miR-27 a)can promote bone regeneration and protect against GC-ONFH to some extent.

关 键 词：miR-27a 外泌体 成骨 股骨头坏死 人脐静脉内皮细胞 基因靶向治疗 

分 类 号：R681.8[医药卫生—骨科学]