骨关节炎患者成骨细胞胞外体miRNAs表达谱的差异  

Differential expression of exosomal miRNAs in osteoblasts in osteoarthritis

作　　者：刘博豪 吴鹏飞[2] 梅林 罗勇[1] 李洪星[1] 毛新展[1] LIU Bohao;WU Pengfei;MEI Lin;LUO Yong;LI Hongxing;MAO Xinzhan(Department of Orthopedics,Second Xiangya Hospital,Central South University,Changsha 410011;Center for Medical Genetics,School of Life Sciences,Central South University,Changsha 410008,China)

机构地区：[1]中南大学湘雅二医院骨科,长沙410011 [2]中南大学生命科学学院,医学遗传学研究中心,长沙410008

出　　处：《中南大学学报（医学版）》2018年第12期1294-1300,共7页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(81371997).

摘　　要：目的:通过分析骨关节炎(osteoarthritis,OA)患者软骨下骨组织中成骨细胞胞外体miRNAs的表达差异,探究OA发生发展过程中可能涉及的关键胞外体miRNAs。方法:共纳入6名膝OA患者,收集其膝关节软骨下骨组织,选取磨损严重侧为OA组,取磨损较少侧为正常对照组。细胞培养后提取成骨细胞胞外体,进行特征鉴定。提取胞外体miRNAs,用高通量测序并筛选两组间表达差异显著者(log2Ratio≥2)进行靶基因预测分析,并进一步行定量反转录PCR分析。结果:提取物经鉴定为胞外体,在扫描电镜下呈囊泡状双层膜结构,直径30～200 nm,体积峰度50～150 nm。高通量测序共筛出5个OA组表达显著上调的miRNAs,差异倍数最大者为hsa-miR-4717-5p,预测靶基因为RGS2;定量反转录PCR亦显示OA组hsa-miR-4717-5p相对表达量较对照组显著增高(2.243 vs 0.480,P<0.01)。结论:OA患者成骨细胞胞外体miRNAs表达谱较正常者有显著差异。部分表达显著上调的miRNAs可能通过代谢相关通路介导OA的发生和发展。Objective: To analyze the diff erentially expressed exosomal miRNAs in subchondral osteoblasts in patients with osteoarthritis (OA) and to investigate the key miRNAs potentially involved in the occurrence and progression of OA. Methods: Subchondral bones were harvested from 6 patients with OA. All subjects were divided into two groups which was based on the severity of joint wear: An OA group, severely worn side of subchondral bone, and a control group, less worn side of subchondral bone. Th e exosomes were extracted from osteoblast cells and their characteristics were identifi ed. Th en exosomal miRNAs were extracted and sequencing analysis was conducted to compare the expression in the two groups. The most differentially expressed ones (log2Ratio≥2) were subject to miRNA target prediction and quantitative reverse transcription PCR (RT-qPCR) to further quantify the difference. Results: Osteoblast extractions were confirmed to be exosomes, which were small doublemembranous vesicles with 30–200 nm in diameter and 50–150 nm in peak value of particle size under the scanning microscope. High-throughput sequencing revealed 124 miRNAs whose expression significantly increased in the OA group. The most differentially expressed one with maximum fold change was hsa-miR-4717-5p and its target gene was RGS2. RT-qPCR demonstrated hsa-miR-4717-5p expression in the OA group was relatively higher than that in the control group (2.243 vs 0.480, P<0.01). Conclusion: There is distinct difference in expression profiles of exosomal miRNAs in subchondral osteoblasts between patients with OA and normal subjects. Up-regulated expression of miRANs might participate in OA occurrance and progression.

关 键 词：骨关节炎 胞外体 MIRNAS 成骨细胞 软骨下骨 

分 类 号：R684.3[医药卫生—骨科学]