抑制环氧合酶-2表达对软骨细胞发育成熟的影响  

Effect of inhibition of cyclooxygenase-2 expression on chondrocyte growth and maturation

作　　者：王灿[1] 司文腾[1] WANG Can;SI Wenteng(Zhengzhou Orthopedics Hospital,Zhengzhou 450052,Henan,China)

机构地区：[1]郑州市骨科医院,河南郑州450052

出　　处：《中医正骨》2022年第11期1-6,共6页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20191147)。

摘　　要：目的:探讨抑制环氧合酶-2(cyclooxygenase-2,COX-2)表达对软骨细胞发育成熟的影响及其作用机制。方法:①分析温度对SV40病毒大T抗原介导永生化小鼠软骨细胞(mouse chondrocytes immortalized by SV40 large Tantigen,MCT)发育成熟的影响。将MCT在温度32℃、CO_(2)浓度8%的细胞培养箱内进行培养,待MCT汇合率至80%时,分别置于32℃下(32℃培养组)和37℃下(37℃培养组)继续培养。培养72h后,采用相差倒置显微镜观察细胞形态学变化;采用CIAS大恒细胞图像分析系统测量细胞长径、短径,并计算细胞体积;提取各复孔中MCT的RNA,逆转录后采用实时定量PCR检测MCT中COX-2、胶原蛋白α-1(X)[Collagenalpha-1(X)chain,COL10A1]的mRNA表达水平;提取各复孔中MCT的总蛋白,采用蛋白质印迹法检测MCT中COX-2、COL10A1的蛋白表达水平。②筛选COX-2抑制剂NS-398的最佳抑制浓度。将MCT在温度32℃、CO_(2)浓度8%的细胞培养箱内进行培养,待MCT汇合率至80%时,按照NS-398终浓度为0.2μmol·L^(-1)、1μmol·L^(-1)、2μmol·L^(-1)、10μmol·L^(-1)、20μmol·L^(-1)、25μmol·L^(-1)、30μmol·L^(-1)、40μmol·L^(-1)、50μmol·L^(-1)、60μmol·L^(-1)将MCT分为10个干预组,分别加入等体积不同浓度的NS-398,空白对照组加入等体积的二甲亚砜。于37℃下培养24h后,提取各复孔中MCT的RNA,逆转录后采用实时定量PCR检测MCT中COX-2的mRNA表达量,比较和筛选NS-398对COX-2表达的最佳抑制浓度。③分析NS-398最佳抑制浓度对MCT发育成熟的影响。以②中NS-398最低浓度为对照,分析NS-398最佳抑制浓度对MCT发育成熟的影响。将MCT在温度32℃、CO_(2)浓度8%的细胞培养箱内进行培养,待MCT汇合率至80%时,按照NS-398终浓度为0.2μmol·L^(-1)、40μmol·L^(-1)将MCT分为0.2μmol·L^(-1)NS-398干预组和40μmol·L^(-1)NS-398干预组,分别加入等体积不同浓度的NS-398,移至温度37℃、CO_(2)浓度8%的细胞培养箱内继续培养。培养72h后,采用①�Objective:To investigate the effect of inhibition of cyclooxygenase-2(COX-2)expression on the growth and maturation of chondrocytes and its mechanism of action.Methods:(1)To analyze the effect of temperature on the growth and maturation of mouse chondrocytes immortalized by SV40 large T antigen(MCTs).MCTs were cultured in a cell incubator at 32 ℃ with 8% CO.When cell confluence reached 80%,cells were divided into two groups and cultured at 32 ℃(32 ℃ culture group)and 37 ℃(37 ℃ culture group),respectively.After 72 h of culture, the morphological changes of the cells were observed by an inverted phase-contrast microscope.The cell image analysis system(CIAS)was used to measure the long diameter and short diameter of cells and calculate the cell volume.The RNAs of MCTs were extracted from duplicated wells, and mRNA expression levels of COX-2 and collagen alpha-1(X)chain(COL10 A1)in MCTs were detected by real-time quantitative PCR after reverse transcription.The total proteins of MCTs were extracted from duplicated wells, and the protein expression levels of COX-2 and COL10 A1 in MCTs were detected by Western blot.(2)To screen the optimal inhibitory concentration of COX-2 inhibitor NS-398.MCTs were cultured in a cell incubator at 32 ℃ with 8% CO.When cell confluence reached 80%,MCTs were divided into 10 groups, added with the same volume of NS-398 with final concentration of 0.2,1,2,10,20,25,30,40,50,and 60 μmol/L.An equal volume of dimethyl sulfoxide was added to the blank control group.After culture at 37 ℃ for 24 h, the RNAs of MCTs were extracted from duplicated wells.The COX-2 mRNA expression in MCTs was detected by real-time quantitative PCR after reverse transcription, and the optimal inhibitory concentration of NS-398 on COX-2 expression was compared and screened out.(3)To analyze the effect of the optimal inhibitory concentration of NS-398 on the growth and maturation of MCTs.The effect of the optimal inhibitory concentration of NS-398 on the growth and maturation of MCTs was analyzed with the

关 键 词：内生软骨瘤病 软骨细胞 环氧合酶2 环氧合酶2抑制剂 

分 类 号：R681.3[医药卫生—骨科学]