骨关节炎软骨损伤中miRNA-214的作用机制及靶向基因研究  

Mechanism and targeted genes of miRNA-214 in osteoarthritis-induced cartilage injury

作　　者：王最 徐佳妮 吴良邦 钱钧[2] WANG Zui;XU Jiani;WU Liangbang;QIAN Jun(The 903rd Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army,Hangzhou 310004,Zhejiang,China;Quzhou People’s Hospital,Quzhou 324000,Zhejiang,China)

机构地区：[1]解放军联勤保障部队第九〇三医院,浙江杭州310004 [2]衢州市人民医院,浙江衢州324000

出　　处：《中医正骨》2023年第3期6-14,30,共10页The Journal of Traditional Chinese Orthopedics and Traumatology

摘　　要：目的:探讨骨关节炎(osteoarthritis,OA)软骨损伤中微小RNA(microRNA,miRNA)-214的作用机制及靶向基因。方法:①OA患者膝关节损伤软骨组织中miRNA-214表达量检测。根据软骨损伤程度Outerbridge分级标准将收集的OA患者膝关节软骨组织进行分级,分别提取各软骨组织的总RNA,逆转录cDNA后采用实时定量PCR检测损伤软骨组织中miRNA-214的表达量。②miRNA-214在OA大鼠软骨损伤中的作用机制分析。采用手术剪断大鼠前交叉韧带的方法建立大鼠OA模型。将40只造模成功的大鼠随机分为miRNA-214高表达组、miRNA-214低表达组、阴性对照组及模型组,将10只接受手术但不剪断前交叉韧带的大鼠纳入假手术组。设计、合成miRNA-214模拟物、miRNA-214抑制剂及miRNA-214阴性对照序列,构建慢病毒表达载体,完成病毒包装。在miRNA-214高表达组、miRNA-214低表达组、阴性对照组、模型组及假手术组大鼠右侧膝关节腔内分别注射100μL miRNA-214模拟物慢病毒混悬液、100μLmiRNA-214抑制剂慢病毒混悬液、100μL miRNA-214阴性对照慢病毒混悬液、100μL生理盐水、100μL生理盐水。干预后4周,采集各组大鼠腹主动脉血,检测血清白细胞介素(interleukin,IL)-17、IL-23水平;制备各组大鼠膝关节软骨组织石蜡切片,HE染色后观察软骨组织病理学改变;采用免疫印迹法检测各组大鼠膝关节软骨组织中聚集蛋白聚糖、Ⅱ型胶原蛋白(collagen Ⅱ,ColⅡ)、基质金属蛋白酶(matrix metalloproteinase,MMP)-13的蛋白相对表达量。③OA软骨损伤相关的miRNA-214靶向基因分析。检索miRNA靶基因数据库中miRNA-214的靶向基因,根据文献资料筛选与骨代谢相关的人类miRNA-214靶向基因。采用双荧光素酶实验验证miRNA-214对活化转录因子4(activating transcription factor 4,ATF4)的靶向性。采用免疫印迹法检测各组大鼠右侧膝关节软骨组织中ATF4的蛋白相对表达量。结果:①OA患者膝关节损�Objective:To investigate the mechanism and targeted genes of micro RNA(miRNA)-214 in osteoarthritis(OA)-induced cartilage injury.Methods:①Deltection of miRNA-214 expression in injured cartilage of the knee joint in patients with OA.The knee cartilage tissues of OA patients were graded according to the Outerbridge classification for cartilage injury.The total RNA of each cartilage tssue was extracted,and the expression of miRNA-214 in the injured cartilage was detected by real-time quantitative PCR after reverse transcription of cDNA.②Analysis of mechanism of miRNA-214 in cartilage injury in OA rats.The OA model was established in rats by the surgical cutting of the anterior cruciate ligament.Forty OA model rats were randomly divided into a miRNA-214 high expression group,a miRNA-214 low expression group,a negative control group,and a model group.Another 10 rats undergoing surgery without cuting the anterior cruciate ligament were ssgned into the sham operation group.The sequences of miRNA-214 mimics,miRNA-214 inhibitors,and miRNA-214 negative control were designed and synthesized,and lentivirus expression veclor was constructed,followed by lentiviral packaging.Rats in the miRNA-214 high expression group,the miRNA-214 low expression group,the negative control group,the model group,and the sham operation group were injected with 100μL of miRNA-214 mimic lentivirus suspension,100μL of miRNA-214 inhibitor lentivirus suspension,100μL of miRNA-214 negative control lentivirus suspension,100μL of normal saline,and 100μL of normal saline,respectively,at the right knee cavity.At four weeks after the intervention,blood in the abdominal aorta of all rats was sampled and serum interleukin(IL)-17 and IL-23 levels were measured.Paraffin sections of knee cartilage tissues were prepared,and the pathological changes in cartilage tissues were observed following HE staining.The relative protein expression levels of aggrecan,collagen Ⅱ(ColⅡ),and matrix metalloproteinase(MMP)-13 in the knee cartilage tissues of rats in ea

关 键 词：骨关节炎 软骨疾病 微RNAS 基因表达 大鼠 动物实验 

分 类 号：R684.3[医药卫生—骨科学]