慢性昼夜节律紊乱对大鼠膝骨关节炎样软骨损伤的机制研究  

Mechanism of chronic circadian rhythm disorder on knee osteoarthritis-like cartilage injury in rats

作　　者：杨春生[1] 陈祢[1] 武恒敏 张铁成[1] 王宝兰[1] YANG Chunsheng;CHEN Mi;WU Hengmin;ZHANG Tiecheng;WANG Baolan(Department of Rehabilitation Medicine,the First Affiliated Hospital of Xinjiang Medical University,Urumqi,Xinjiang 830000,P.R.China)

机构地区：[1]新疆医科大学第一附属医院康复医学科,乌鲁木齐830000

出　　处：《华西医学》2024年第6期891-898,共8页West China Medical Journal

基　　金：康复医学四川省重点实验室开放课题(KFYXSZDSYS-10);新疆医科大学第一附属医院青年科研起航专项基金(2022YFY-QKQN-32)。

摘　　要：目的探讨昼夜节律紊乱对大鼠膝关节软骨的影响及生物钟基因基本螺旋体型蛋白1(basic helixloop-helix ARNT like 1,Bmal1)对细胞周期相关基因调控的机制。方法将40只大鼠随机分为正常组、昼夜节奏紊乱组(紊乱组)、Bmal1过表达慢病毒感染的昼夜节律紊乱组(Bmal1上调组)和Bmal1过表达慢病毒阴性感染的昼夜节律紊乱组(Bmal1阴性感染组),每组10只。采用番红固绿染色、原位末端转移酶标记法染色、免疫组织化学染色、逆转录聚合酶链反应和蛋白质印迹技术,比较不同组别软骨组织病理变化,软骨细胞凋亡情况,Bmal1、WEE1细胞周期G2检测点激酶(WEE1 G2 checkpoint kinase,Wee1)、细胞周期依赖性激酶1(cyclindependent kinase 1,Cdk1)、细胞周期蛋白B1(cyclin B1,Ccnb1)、BCL2相关X蛋白(BCL2-associated X protein,Bax)、细胞凋亡调节剂2(apoptosis regulator 2,Bcl2)、白细胞介素1(interleukin 1,Il1)、白细胞介素6(interleukin 6,Il6)、肿瘤坏死因子(tumor necrosis factor,Tnf)和基质金属蛋白酶13(matrix metallopeptidase 13,Mmp13)基因的mRNA相对表达量,以及BMAL1、WEE1、CDK1、CCNB1、BAX和BCL2蛋白相对表达水平,根据mRNA相对表达量,进行相关性分析。结果番红固绿染色见正常组软骨基质厚度正常,呈均匀的红色;紊乱组和Bmal1阴性感染组软骨基质破坏,蛋白聚糖丢失明显,呈不均匀红色;Bmal1上调组软骨基质厚度基本正常,蛋白聚糖无明显丢失,红色略欠均匀。与正常组相比,紊乱组和Bmal1阴性感染组关节软骨中凋亡细胞的阳性率升高,Bmal1、Wee1、Bcl2的mRNA及对应蛋白相对表达量下调,Cdk1、Ccnb1、Bax的mRNA和对应蛋白相对表达量上调,Il1、Il6、Tnf、Mmp13的mRNA相对表达量上调,差异有统计学意义(P<0.05);Bmal1上调组关节软骨中凋亡细胞阳性率无明显变化,Bmal1、Wee1、Bcl2、Cdk1、Ccnb1、Bax的mRNA及对应蛋白相对表达量,及Il1、Il6、Tnf、Mmp13的mRNA相对表达量差异无统计Objective To investigate the effect of circadian rhythm disorder on rat knee cartilage and the mechanism of basic helix-loop-helix ARNT like 1(Bmal1)on the regulation of cell cycle-related genes.Methods Forty rats were randomly divided into normal group,circadian rhythm disorder group(disorder group),Bmal1 overexpression lentivirus infection circadian rhythm disorder group(Bmal1 up-regulated group)and Bmal1 overexpression lentivirus negative infection circadian rhythm disorder group(Bmal1 negative infection group),with 10 rats in each group.Saffron fast green staining,TdT-mediated dUTP nick-end labeling staining,immunohistochemical staining,reverse transcription polymerase chain reaction and Western blotting were used to compare the pathological changes of cartilage tissue,the apoptosis of chondrocytes,and the relative mRNA expression levels of Bmal1,WEE1 G2 checkpoint kinase(Wee1),cyclindependent kinase 1(Cdk1),cyclin B1(Ccnb1),BCL2-associated X protein(Bax),apoptosis regulator 2(Bcl2),interleukin 1(Il1),interleukin 6(Il6),tumor necrosis factor(Tnf)and matrix metallopeptidase 13(Mmp13)among different groups.The relative expression levels of BMAL1,WEE1,CDK1,CCNB1,BAX and BCL2 proteins were detected,and correlation analysis was performed according to the relative expression of mRNA.Results Safranine fast green staining showed that the thickness of cartilage matrix in the normal group was normal and uniform red.The cartilage matrix in the disorder group and the Bmal1 negative infection group was destroyed,and the proteoglycan was lost obviously,showing uneven red.The thickness of cartilage matrix in the Bmal1 up-regulated group was basically normal,and the proteoglycan was not lost obviously,and the red was slightly less uniform.Compared with those of the normal group,the positive rates of apoptotic cells in articular cartilage of the disorder group and the Bmal1 negative infection group increased significantly,the mRNA and protein expression levels of Bmal1,Wee1,and Bcl2 were down-regulated,the mRNA and protein ex

关 键 词：昼夜节律 生物钟 Bmal1基因 细胞周期 骨关节炎 大鼠 

分 类 号：R684.3[医药卫生—骨科学]