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[1]王燦,司文騰.抑制環(huán)氧合酶-2表達(dá)對(duì)軟骨細(xì)胞發(fā)育成熟的影響[J].中醫(yī)正骨,2022,34(11):1-6.
　WANG Can,SI Wenteng.Effect of inhibition of cyclooxygenase-2 expression on chondrocyte growth and maturation[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2022,34(11):1-6.
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抑制環(huán)氧合酶-2表達(dá)對(duì)軟骨細(xì)胞發(fā)育成熟的影響()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第34卷
期數(shù):
2022年11期
頁(yè)碼:
1-6
欄目:
基礎(chǔ)研究
出版日期:
2022-11-11

文章信息/Info

Title:
Effect of inhibition of cyclooxygenase-2 expression on chondrocyte growth and maturation
作者:
王燦; 司文騰
(鄭州市骨科醫(yī)院,河南 鄭州 450052)
Author(s):
WANG Can; SI Wenteng
Zhengzhou Orthopedics Hospital,Zhengzhou 450052,Henan,China
關(guān)鍵詞:
內(nèi)生軟骨瘤病;  軟骨細(xì)胞;  環(huán)氧合酶2;  環(huán)氧合酶2抑制劑
Keywords:
enchondromatosis;  chondrocytes;  cyclooxygenase 2;  cyclooxygenase 2 inhibitors
摘要:
目的:探討抑制環(huán)氧合酶-2(cyclooxygenase-2,COX-2)表達(dá)對(duì)軟骨細(xì)胞發(fā)育成熟的影響及其作用機(jī)制。方法:①分析溫度對(duì)SV40病毒大T抗原介導(dǎo)永生化小鼠軟骨細(xì)胞(mouse chondrocytes immortalized by SV40 large T antigen,MCT)發(fā)育成熟的影響。將MCT在溫度32 ℃、CO2濃度8%的細(xì)胞培養(yǎng)箱內(nèi)進(jìn)行培養(yǎng),待MCT匯合率至80%時(shí),分別置于32 ℃下(32 ℃培養(yǎng)組)和37 ℃下(37 ℃培養(yǎng)組)繼續(xù)培養(yǎng)。培養(yǎng)72 h后,采用相差倒置顯微鏡觀察細(xì)胞形態(tài)學(xué)變化; 采用CIAS大恒細(xì)胞圖像分析系統(tǒng)測(cè)量細(xì)胞長(zhǎng)徑、短徑,并計(jì)算細(xì)胞體積; 提取各復(fù)孔中MCT的RNA,逆轉(zhuǎn)錄后采用實(shí)時(shí)定量PCR檢測(cè)MCT中COX-2、膠原蛋白α-1(X)[Collagen alpha-1(X)chain,COL10A1]的mRNA表達(dá)水平; 提取各復(fù)孔中MCT的總蛋白,采用蛋白質(zhì)印跡法檢測(cè)MCT中COX-2、COL10A1的蛋白表達(dá)水平。②篩選COX-2抑制劑NS-398的最佳抑制濃度。將MCT在溫度32 ℃、CO2濃度8%的細(xì)胞培養(yǎng)箱內(nèi)進(jìn)行培養(yǎng),待MCT匯合率至80%時(shí),按照NS-398終濃度為0.2 μmol·L-1、1 μmol·L-1、2 μmol·L-1、10 μmol·L-1、20 μmol·L-1、25 μmol·L-1、30 μmol·L-1、40 μmol·L-1、50 μmol·L-1、60 μmol·L-1將MCT分為10個(gè)干預(yù)組,分別加入等體積不同濃度的NS-398,空白對(duì)照組加入等體積的二甲亞砜。于37 ℃下培養(yǎng)24 h后,提取各復(fù)孔中MCT的RNA,逆轉(zhuǎn)錄后采用實(shí)時(shí)定量PCR檢測(cè)MCT中COX-2的mRNA表達(dá)量,比較和篩選NS-398對(duì)COX-2表達(dá)的最佳抑制濃度。③分析NS-398最佳抑制濃度對(duì)MCT發(fā)育成熟的影響。以②中NS-398最低濃度為對(duì)照,分析NS-398最佳抑制濃度對(duì)MCT發(fā)育成熟的影響。將MCT在溫度32 ℃、CO2濃度8%的細(xì)胞培養(yǎng)箱內(nèi)進(jìn)行培養(yǎng),待MCT匯合率至80%時(shí),按照NS-398終濃度為0.2 μmol·L-1、40 μmol·L-1將MCT分為0.2 μmol·L-1 NS-398干預(yù)組和40 μmol·L-1 NS-398干預(yù)組,分別加入等體積不同濃度的NS-398,移至溫度37 ℃、CO2濃度8%的細(xì)胞培養(yǎng)箱內(nèi)繼續(xù)培養(yǎng)。培養(yǎng)72 h后,采用①中方法測(cè)量細(xì)胞體積,檢測(cè)MCT中COX-2、COL10A1的mRNA和蛋白表達(dá)水平。采用Person相關(guān)分析法分析COX-2 mRNA表達(dá)量、COL10A1 mRNA表達(dá)量與MCT細(xì)胞體積的相關(guān)性。結(jié)果:①溫度對(duì)MCT發(fā)育成熟的影響。32 ℃培養(yǎng)組MCT表現(xiàn)為持續(xù)增殖狀態(tài),37 ℃培養(yǎng)組MCT表現(xiàn)為肥大狀態(tài)。37 ℃培養(yǎng)組MCT細(xì)胞體積大于32 ℃培養(yǎng)組[(2 336.19±24.69)μm3,(1 195.27±13.28)μm3,t=70.488,P=0.000],COX-2、COL10A1的mRNA和蛋白表達(dá)量均高于32 ℃培養(yǎng)組(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抑制劑NS-398的最佳抑制濃度。37 ℃下培養(yǎng)24 h后,11組MCT中COX-2的mRNA表達(dá)量比較,差異有統(tǒng)計(jì)學(xué)意義(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)。40 μmol·L-1 NS-398干預(yù)組MCT中COX-2的mRNA表達(dá)量低于對(duì)照組和其他濃度NS-398干預(yù)組(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最佳抑制濃度對(duì)MCT發(fā)育成熟影響的分析結(jié)果。培養(yǎng)72 h后,40 μmol·L-1 NS-398干預(yù)組MCT細(xì)胞體積小于0.2 μmol·L-1 NS-398干預(yù)組[(995.64±10.98)μm3,(2 011.07±20.52)μm3,t=70.488,P=0.000],COX-2、COL10A1的mRNA和蛋白表達(dá)量均低于0.2 μmol·L-1 NS-398干預(yù)組(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相關(guān)分析結(jié)果表明,COX-2 mRNA表達(dá)量與COL10A1 mRNA表達(dá)量、COX-2 mRNA表達(dá)量與MCT細(xì)胞體積、COL10A1 mRNA表達(dá)量與MCT細(xì)胞體積均呈正相關(guān)(r=0.552,P=0.011; r=0.658,P=0.001; r=0.590,P=0.003)。結(jié)論:抑制COX-2表達(dá)能夠抑制軟骨細(xì)胞發(fā)育成熟,其作用機(jī)制與COL10A1表達(dá)下調(diào)有關(guān)。
Abstract:
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:①To 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% CO2.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(COL10A1)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 COL10A1 in MCTs were detected by Western blot.②To screen the optimal inhibitory concentration of COX-2 inhibitor NS-398.MCTs were cultured in a cell incubator at 32 ℃ with 8% CO2.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.③To 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% CO2.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% CO2.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 COL10A1 in MCTs were detected.The correlation between mRNA expression levels of COX-2 and COL10A1 and MCT volume was analyzed by Person correlation analysis.Results:①Effect 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 COL10A1 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; COL10A1:2.32±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).②Optimal 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).③Effect 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 COL10A1 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; COL10A1:0.49±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 COL10A1 mRNA expression and MCT volume,and COL10A1 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 COL10A1 expression.

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備注/Memo

備注/Memo:
基金項(xiàng)目:河南省醫(yī)學(xué)科技攻關(guān)計(jì)劃聯(lián)合共建項(xiàng)目(LHGJ20191147)
更新日期/Last Update: 1900-01-01