针刀治疗膝骨关节炎大鼠的转录组测序及实验验证  

Transcriptional profiling and experimental validation of acupotomy for knee osteoarthritis in rats

作　　者：刘彦彤 王世轩[2] 赵双利[3] 魏巍 王东海[2] 姜宗坤 刘洪飞 Liu Yantong;Wang Shixuan;Zhao Shuangli;Wei Wei;Wang Donghai;Jiang Zongkun;Liu Hongfei(Liaoning University of Traditional Chinese Medicine,Shenyang 110032,Liaoning Province,China;Department of Orthopedics I,the Second Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Shenyang 110034,Liaoning Province,China;Department of Orthopedics IV,the Second Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Shenyang 110034,Liaoning Province,China;Department of Orthopedics II,the Second Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Shenyang 110034,Liaoning Province,China)

机构地区：[1]辽宁中医药大学,辽宁省沈阳市110032 [2]辽宁中医药大学附属第二医院骨伤一科,辽宁省沈阳市110034 [3]辽宁中医药大学附属第二医院骨伤四科,辽宁省沈阳市110034 [4]辽宁中医药大学附属第二医院骨伤二科,辽宁省沈阳市110034

出　　处：《中国组织工程研究》2025年第20期4239-4248,共10页Chinese Journal of Tissue Engineering Research

基　　金：国家中医药管理局全国名老中医药专家传承工作室建设项目(国中医药人教函[2022]75号);项目负责人:赵双利。

摘　　要：背景:针刀治疗膝骨关节炎临床疗效确切,但针刀干预膝骨关节炎在转录组水平上的调节机制仍未明确。目的:通过针刀干预骨关节炎大鼠,对软骨样本进行转录组测序和生物信息学分析并加以验证,揭示针刀干预骨关节炎大鼠的作用机制。方法:采用随机数字表法将48只SD大鼠随机分为针刀组、模型组和假手术组,每组16只。针刀组、模型组采用内侧半月板失稳术建立骨关节炎模型,造模6周后针刀组大鼠每周进行1次针刀治疗,共治疗4周。治疗4周后,对各组大鼠膝关节进行Micro CT扫描,对关节软骨进行苏木精-伊红染色、番红O-固绿染色及Mankin评分,采用Elisa法检测血清炎症因子水平,对软骨样本进行转录组测序,采用R软件进行差异基因生物信息学分析,采用蛋白互作网络和Cytoscape软件筛选核心靶点并进行RT-qPCR验证。结果与结论:①相较于假手术组,模型组大鼠关节软骨表面粗糙不平、关节间隙狭窄,关节表层结构破坏,Mankin评分升高,血清白细胞介素6、肿瘤坏死因子α、基质金属蛋白酶13水平显著上升(P<0.05);与模型组相比,针刀组大鼠关节软骨表面较为光滑,关节间隙增宽,关节表面轻微不规则,Mankin评分降低,血清白细胞介素6、肿瘤坏死因子α、基质金属蛋白酶13水平显著降低(P<0.05)。②基因本体论及京都基因组和基因组百科全书分析涉及蛋白分解代谢、自噬、丝裂原活化蛋白激酶、核因子kB及Wnt信号通路等,蛋白互作网络及Cytoscape筛选出共济失调毛细血管扩张突变、类Myb SWIRM和MPN结构域蛋白1、热休克蛋白90α1、NIPBL粘连蛋白负载因子4个关键基因。③模型组大鼠关节软骨中共济失调毛细血管扩张突变、类Myb SWIRM和MPN结构域蛋白1、热休克蛋白90α1、NIPBL粘连蛋白负载因子mRNA表达均低于假手术组(P<0.05),针刀组大鼠关节软骨中共济失调毛细血管扩张突变、类Myb SWIRM和MPNBACKGROUND:The regulatory mechanisms of acupotomy intervention for knee osteoarthritis at a transcriptome level are not well understood despite its proven clinical efficacy.OBJECTIVE:Using acupotomy therapy in a rat model of knee osteoarthritis,to conduct transcriptome sequencing and bioinformatics analysis on cartilage samples,along with validation,and to reveal the molecular regulatory mechanisms involved in this therapy for knee osteoarthritis in rats.METHODS:Forty-eight Sprague-Dawley rats were selected and divided into three groups by random number table method,acupotomy group,model group,and sham operation group,with 16 rats in each group.Osteoarthritis models were induced by medial meniscus instability in the acupotomy group and model group.After successful modeling,acupotomy group rats were treated with acupotomy once a week,for 4 weeks in total.After the intervention,cartilage samples from the rat’s knee joint were stained with hematoxylin-eosin staining and safranin O-fast green staining,evaluated by Mankin scores,and underwent MicroCT scanning.Serum inflammatory factor levels were detected by Elisa.Transcriptome sequencing was performed on the remaining cartilage samples,and the data were analyzed using R software to identify differential gene expression levels among the groups.Core targets were screened through protein-protein interaction network and Cytoscape and validated using RT-qPCR.RESULTS AND CONCLUSION:Compared with the sham operation group,rats in the model group had rough and uneven articular cartilage surfaces,narrowed joint spaces,destroyed articular surface structures,elevated Mankin scores,and significant increases in serum levels of interleukin-6,tumor necrosis factor-α,and matrix metalloproteinase 13(P<0.05).Compared with the model group,rats in the acupotomy group had smoother articular cartilage surfaces,wider joint spaces,slightly irregular articular surfaces,lower Mankin scores,and significantly lower serum levels of interleukin-6,tumor necrosis factor-α,and matrix metalloprote

关 键 词：膝骨关节炎 针刀 骨关节炎模型 内侧半月板韧带失稳术 转录组测序 差异基因 动物实验 实验验证 工程化组织构建 

分 类 号：R459.9[医药卫生—治疗学] R319[医药卫生—临床医学] R245.3