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中方法测量细胞体积,检测MCT中COX-2、COL10A1的mRNA和蛋白表达水平。采用Person相关分析法分析COX-2mRNA表达量、COL10A1mRNA表达量与MCT细胞体积的相关性。结果:①温度对MCT发育成熟的影响。32℃培养组MCT表现为持续增殖状态,37℃培养组MCT表现为肥大状态。37℃培养组MCT细胞体积大于32℃培养组[(2336.19±24.69)μm3,(1195.27±13.28)μm3,t=70.488,P=0.000],COX-2、COL10A1的mRNA和蛋白表达量均高于32℃培养组(COX-2:2.09±0.26,1.33±0.35,t=3.019,P=0.039;2.37±0.26,1.58±0.19,t=4.249,P=0.013;COL10A1:2.32±0.34,1.41±0.21,t=3.944,P=0.017;2.56±0.28,1.59±0.17,t=5.129,P=0.007)。②COX-2抑制剂NS-398的最佳抑制浓度。37℃下培养24h后,11组MCT中COX-2的mRNA表达量比较,差异有统计学意义(1.00±0.11,0.89±0.09,0.81±0.08,1.24±0.13,0.63±0.06,0.69±0.07,0.71±0.07,0.70±0.06,0.43±0.04,0.85±0.09,0.84±0.08,F=38.074,P=0.000)。40μmol·L^(-1)NS-398干预组MCT中COX-2的mRNA表达量低于对照组和其他浓度NS-398干预组(P=0.001,P=0.001,P=0.001,P=0.001,P=0.002,P=0.001,P=0.001,P=0.001,P=0.001,P=0.001)。③NS-398最佳抑制浓度对MCT发育成熟影响的分析结果。培养72h后,40μmol·L^(-1)NS-398干预组MCT细胞体积小于0.2μmol·L^(-1) NS-398干预组[(995.64±10.98)μm3,(2011.07±20.52)μm3,t=70.488,P=0.000],COX-2、COL10A1的mRNA和蛋白表达量均低于0.2μmol·L^(-1) NS-398干预组(COX-2:0.39±0.04,0.99±0.09,t=15.988,P=0.000;1.53±0.16,3.97±0.40,t=9.810,P=0.000;COL10A1:0.49±0.05,1.01±0.11,t=11.258,P=0.000;1.09±0.12,2.74±0.29,t=9.106,P=0.001)。Pearson相关分析结果表明,COX-2mRNA表达量与COL10A1mRNA表达量、COX-2mRNA表达量与MCT细胞体积、COL10A1mRNA表达量与MCT细胞体积均呈正相关(r=0.552,P=0.011;r=0.658,P=0.001;r=0.590,P=0.003)。结论:抑制COX-2表达能够抑制软骨细胞发育成熟,其作用机制与COL10A1表达下调有关。
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 lowest concentration of NS-398 in(2) as the control.MCTs were cultured in a cell incubator at 32 ℃ with 8% CO.When cell confluence reached 80%,MCTs were divided into 0.2 μmol/L NS-398 intervention group and 40 μmol/L NS-398 intervention group with the same volume of NS-398 at corresponding concentrations added.Subsequently, MCTs continued to culture in an incubator at 37 ℃ with 8% CO.After culture for 72 h, cell volume was measured by the method as described in(1),and mRNA and protein expression levels of COX-2 and COL10 A1 in MCTs were detected.The correlation between mRNA expression levels of COX-2 and COL10 A1 and MCT volume was analyzed by Person correlation analysis.Results:(1)Effect of temperature on the growth and maturation of MCTs.The MCTs in the 32 ℃ culture group showed sustained proliferation, while those in the 37 ℃ culture group showed hypertrophy.The volume of MCTs in the 37 ℃ culture group was larger than that in the 32 ℃ culture group(2 336.19±24.69 vs 1 195.27±13.28 μm(3),t=70.488,P=0.000),and the mRNA and protein expression of COX-2 and COL10 A1 was higher than that in the 32 ℃ culture group(COX-2:2.09±0.26 vs 1.33±0.35,t=3.019,P=0.039;2.37±0.26 vs 1.58±0.19,t=4.249,P=0.013;COL10 A1:2.32±0.34 vs 1.41±0.21,t=3.944,P=0.017;2.56±0.28 vs 1.59±0.17,t=5.129,P=0.007).(2)Optimal inhibitory concentration of COX-2 inhibitor NS-398.After culture at 37 ℃ for 24 h, there were significant differences in the mRNA expression of COX-2 between the 11 MCT groups(1.00±0.11,0.89±0.09,0.81±0.08,1.24±0.13,0.63±0.06,0.69±0.07,0.71±0.07,0.70±0.06,0.43±0.04,0.85±0.09,0.84±0.08,F=38.074,P=0.000).The mRNA expression of COX-2 in MCTs of the 40 μmol/L NS-398 intervention group was lower than that of the blank control group and other NS-398 intervention groups(P=0.001,P=0.001,P=0.001,P=0.001,P=0.002,P=0.001,P=0.001,P=0.001,P=0.001,P=0.001).(3)Effect of the optimal inhibitory concentration of NS-398 on MCT growth and maturation.After 72 h of culture, the MCT volume in the 40 μmol/L NS-398 intervention group was smaller than that in the 0.2 μmol/L NS-398 intervention group(995.64±10.98 vs 2 011.07±20.52 μm(3),t=70.488,P=0.000),and the mRNA and protein expression of COX-2 and COL10 A1 was lower than that in the 0.2 μmol/L NS-398 intervention group(COX-2:0.39±0.04 vs 0.99±0.09,t=15.988,P=0.000;1.53±0.16 vs 3.97±0.40,t=9.810,P=0.000;COL10 A1:0.49±0.05 vs 1.01±0.11,t=11.258,P=0.000;1.09±0.12 vs 2.74±0.29,t=9.106,P=0.001).The results of Pearson correlation analysis showed that COX-2 mRNA expression was positively correlated with COL10 A1 mRNA expression and MCT volume, and COL10 A1 mRNA expression was positively correlated with MCT volume(r=0.552,P=0.011;r=0.658,P=0.001;r=0.590,P=0.003).Conclusion:The inhibition of COX-2 expression can inhibit the growth and maturation of chondrocytes, and the underlying mechanism is attributed to the down-regulation of COL10 A1 expression.
作者
王灿
司文腾
WANG Can;SI Wenteng(Zhengzhou Orthopedics Hospital,Zhengzhou 450052,Henan,China)
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)。
