肿瘤坏死因子-α对关节软骨细胞外基质降解代谢的影响

Effect of tumor necrosis factor -α on the catabolism of chondrocyte extracellular matrix

目的观察肿瘤坏死因子α（TNF-α）对关节软骨细胞外基质降解代谢的影响。方法体外分离培养C57BL／6J小鼠关节软骨细胞，实验用第2代细胞，随机分为4组，第1组为空白对照组，第2～4组均加入10μg／LTNF-α刺激，作用时间分别为24、48、72h。采用倒置相差显微镜对各组软骨细胞进行形态学观察，免疫组织化学法对细胞进行染色与鉴定，噻唑蓝（M1Tr）比色法检测各组软骨细胞的增殖活力，实时定量聚合酶链反应（Real-timePCR）检测各组软骨细胞内目的基因基质金属蛋白酶-13（MMP-13）、固醇调节元件结合蛋白-2（SREBP-2）、蛋白聚糖（aggrecan）、Ⅱ型胶原（collagenⅡ）的mRNA表达。结果倒置相差显微镜观察可见，正常软骨细胞向圆形、多角形转化；免疫组织化学染色显示第2代软骨细胞collagenII染色胞质呈明显棕黄色。MTY检测结果显示，与空白对照组比较，各时间段的TNF-α刺激组吸光度（A）值明显降低（P〈0．01），且随时间的延长而降低明显（24h：86．7％，48h：78．0％，72h：73．5％）。Real．timePCR结果表明，与空白对照组比较，各TNF-α刺激组的MMP-13及SREBP-2mRNA的表达量均上调，且随时间延长而升高f24h：0．142±0．073、1．555±0．087（P〈0．05）；48h：0．178±0．005、1．917±0．153（P〈0．01）；72h：0．197±0．062、2．187±0．135（P〈0．01）]aggrecan、collagenIImRNA表达量则随时间延长而降低[24h：0．689±0．064、0．567±0．038（P〈0．05）；48h：0．620±0．049（P〈0．01）、0．304±0．029（P〈0．05）：72h：0．114±0．078、0．092±0．009（P〈0．01）]。结论TNF-α能诱导软骨细胞产生MMP-13，破坏细胞外基质代谢的平衡，促进降解代谢作用。此外，在此过程中SREBP．2的表达与软骨关键基因的表达呈负相关。

Objective To observe the effect of tumor necrosis factor -α （TNF -α） on the catab- olism of chondrocrie extracetlular matrix. Methods Articular cartilage obtained from C57BL/6J mice was digested and cultured in vitro. After the second passage, cells were randomly divided into four groups： group 1 （control group） , and three experimental groups treated with 10 /.Lg/L TNF-α for 24, 48 and 72 h respectively. Inverted microscope was employed to observe the cells and immunocriochemistry staining was used to identify chondrocyte. The proliferation of each group was measured by thiazolyl blue （MTT） colori- merry. Rreal - time quantitative polymerase chain reaction （ Real - time PCR） was used to detect the mRNA expression of matrix metalloproteinase - 13 （ MMP - 13 ） , sterol - regulatory element binding pro- tein （SREBP） - 2, aggrecan and collagen II. Restflts After consecutive passages, the primary chondro- cries had transformed into circle or polygonal shapes. Immunocytochemistry staining showed the second passage chondrocytes which cytoplasm was stained claybank were positive for anti - collagen ]I antibody. MT1~ assay demonstrated that the absorbance （A） value was significantly lower in each TNF -α experimen- tal group than in the control group （24 h： 86.7% , 48 h： 78.0% , 72 h： 73.5% ）. Real - time PCR results showed that the expression of MMP - 13 and SREBP - 2 mRNA was significantly increased （ P 〈 0. 01 ） in each TNF -α experimental group as compared with the control group and increased with time [24h： 0.142-±0.073, 1.555±0.087 （P〈0.05）;48h：0.178±-0.005, 1.917±0.153 （P〈0.01）; 72 h： 0. 197 ±0. 062, 2. 187 ±0. 135 （P 〈0. 01 ） ]. On the contrary, the expression of aggrecan and colla- gen 1I mRNA was decreased in each TNF - α experimental group and decreased with time [ 24 h ： 0. 689 ± 0.064, 0.567±0.038 （P〈0.05） ; 48 h： 0.620±0.049 （P〈0. 01）, 0.304±0.029 （P〈0.05） ; 72 h： 0. 114 ± 0. 078, 0. 092 ± 0. 009 （ P 〈 0.01 ） ]. Conclusion TNF -α could induce chondrocyte to produce MMP - 13, which leads to break the balance of the extracellular matrix metabolism and promotes the catab- olism. In addition, the expression of SREBP - 2 showed a negative relationship with key cartilage genes during this process.