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慢病毒介导的微小RNA-124基因修饰的骨髓间充质干细胞移植促进大鼠脊髓损伤的修复

Lentivirus-mediated microRNA-124 gene-modified bone marrow mesenchymal stem cell transplantation promotes the repair of spinal cord injury in rats.

作者信息

Song Jia-Lin, Zheng Wei, Chen Wei, Qian Yun, Ouyang Yuan-Ming, Fan Cun-Yi

机构信息

Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.

Department of Orthopedics, The East Campus of Shanghai Sixth People's Hospital, Shanghai University of Medical and Health, Shanghai, China.

出版信息

Exp Mol Med. 2017 May 19;49(5):e332. doi: 10.1038/emm.2017.48.

Abstract

Our study aims to explore the effects of lentivirus-mediated microRNA-124 (miR-124) gene-modified bone marrow mesenchymal stem cell (BMSC) transplantation on the repair of spinal cord injury (SCI) in rats. BMSCs were isolated from the bone marrow of rats. The target gene miR-124 was identified using a luciferase-reporter gene assay. Seventy-two rats were selected for construction of the SCI model, and the rats were randomly divided into the blank group, sham group, SCI group, negative control (NC) group, overexpressed miR-124 group and si-PDXK group. The mRNA expression of miR-124 and the mRNA and protein expression of pyridoxal kinase (PDXK) were detected by quantitative real-time polymerase chain reaction and western blotting. The locomotor capacity of the rats was evaluated using the Basso, Beattie and Bresnahan (BBB) scale. Brdu, neuron-specific enolase (NSE), neurofilament (NF) and microtubule-associated protein 2 (MAP2) were detected using immunohistochemistry. The expression levels of thyrotropin-releasing hormone (TRH), prostacyclin (PGI2) and gangliosides (GM) were measured using an enzyme-linked immunosorbent assay. PDXK was identified as the target gene of miR-124. The overexpressed miR-124 group exhibited higher miR-124 expression than the SCI, NC and si-PDXK groups. Compared with the SCI and NC groups, the PDXK expression was downregulated in the overexpressed miR-124 and si-PDXK groups, and the BBB scores were significantly increased 7, 21 and 35 days after transplantation. The double-labeled positive cell densities (Brdu+NSE/NF/MAP2) and the expression levels of TRH, PGI2 and GM in the overexpressed miR-124 group were significantly higher than those in the NC and SCI groups. These results indicated that miR-124 targeted PDXK to accelerate the differentiation of BMSCs into neurocytes and promote SCI repair.

摘要

我们的研究旨在探讨慢病毒介导的微小RNA-124(miR-124)基因修饰的骨髓间充质干细胞(BMSC)移植对大鼠脊髓损伤(SCI)修复的影响。从大鼠骨髓中分离出BMSC。使用荧光素酶报告基因测定法鉴定靶基因miR-124。选择72只大鼠构建SCI模型,并将大鼠随机分为空白组、假手术组、SCI组、阴性对照组(NC)、miR-124过表达组和si-PDXK组。通过定量实时聚合酶链反应和蛋白质印迹法检测miR-124的mRNA表达以及吡哆醛激酶(PDXK)的mRNA和蛋白质表达。使用Basso、Beattie和Bresnahan(BBB)量表评估大鼠的运动能力。使用免疫组织化学法检测5-溴脱氧尿嘧啶核苷(Brdu)、神经元特异性烯醇化酶(NSE)、神经丝(NF)和微管相关蛋白2(MAP2)。使用酶联免疫吸附测定法测量促甲状腺激素释放激素(TRH)、前列环素(PGI2)和神经节苷脂(GM)的表达水平。PDXK被鉴定为miR-124的靶基因。miR-124过表达组的miR-124表达高于SCI组、NC组和si-PDXK组。与SCI组和NC组相比,miR-124过表达组和si-PDXK组的PDXK表达下调,移植后7天、21天和35天BBB评分显著增加。miR-124过表达组的双标阳性细胞密度(Brdu+NSE/NF/MAP2)以及TRH、PGI2和GM的表达水平显著高于NC组和SCI组。这些结果表明,miR-124靶向PDXK以加速BMSC向神经细胞的分化并促进SCI修复。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7e03/5454445/0a0cb1a2f4c5/emm201748f1.jpg

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