MMP-2、MMP-3、MMP-9、TIMP-3及Col-Ⅱ因子在兔软骨细胞损伤后的表达变化

The Expression Changes of MMP-2, MMP-3, MMP-9, TIMP-3 and Col-Ⅱ mRNA in Rabbit Cartilage Cells after Injury

检测MMP-2、MMP-3、MMP-9、TIMP-3及Ⅱ型胶原细胞外基质因子在正常兔关节软骨细胞和划伤后不同时间点的表达变化。在无菌条件下获取兔膝关节软骨细胞,原代体外培养软骨细胞,六孔板内制备细胞损伤模型。用显微镜观察正常细胞和损伤后1 d、3 d和7 d 3个时间点的软骨细胞增殖情况。采用实时PCR检测正常软骨细胞和损伤后1 d、3 d和7 d 3个时间点的基质金属蛋白酶-2、3和9,基质金属蛋白酶抑制物-3及Ⅱ型胶原的m RNA的表达水平。成功分离软骨,经原代培养后成功建立损伤细胞模型。MMP-2损伤后第1天与正常组相比升高,第3天时下降,第7天明显水平最高。与正常组相比,MMP-3在细胞损伤后第1天表达明显下降,随后第3天逐渐下降,第7天与第3天未见明显变化。MMP-9划伤后第1天比正常组升高,第3天上升至最高水平,第7天下降。TIMP-3基因在损伤后第1天与正常组相比明显下降,随后第3天稍有升高,第7天水平最低。Ⅱ型胶原划伤后第1天与正常组相比明显升高,随后第3天呈下降趋势接近于正常组,第7天又呈上升趋势。MMP-2、MMP-3、MMP-9、TIMP-3因子和Ⅱ型胶原在细胞损伤后的不同时间点有不同的表达行为,这可为调节细胞外基质基因治疗关节软骨损伤选择合适的靶基因和时间窗提供实验依据。

To explore the gene expression levels of matrix metalloproteinase -2, -3 and -9 （MMP-2, -3 and -9）, matrix metalloproteinase inhibitor-3 （TIMP-3） and type II collagen in normal rabbit articular cartilage cells. The rabbit knee joint cartilage cells were obtained under aseptic conditions and cultured in vitro, and the scratch-wound model was established in the six-well plates. The proliferation of chondrocytes in normal cartilage cells and injured cartilage cells that 1, 3 and 7 days after injury were observed under the microscope. The mRNA expressionlevelsof the MMP-2, -3 and -9, TIMP-3 and type II collagen in normal cartilage cells and injured cartilage cells that 1, 3 and 7 days after irtjury were detected by real time PCR.The cartilage was successfully separated, and the injury cell model was successfully established after primary culture of cartilage cells. Compared with the normal group, the gene expression of MMP-2 increased in the first day after injury, but it began to decrease in the third day after injury, and it reached the highest level in the seventh day after injury. Compared with the normal group, the gene expression of MMP-3 increased significantly in the first day after injury, but it gradually decreased in the third day after injury, and there was no obvious difference of MMP-3 level between the third day and the seventh day after injury. The MMP-9 gene expression increased significantly in the first day after injury, which reached the highest level in the third day after injury, then began to decrease in the seventh day after injury. The TIMP-3 gene expression decreased significantly in the first day after injury compared with the normal group, followed by a slight increase in the third day after injury, and reached the lowest level in the seventh day. The gene expression of type II collagen increased significantly in the first day after injury, and it almost reached the normal level in the third day after injury, while showed the upward trend in the seventh day after injury. The expression patterns of MMP-2, -3, -9, TIMP-3 and Col- II mRNA were different at different time points after articular chondrocyte injury, which could provide experimental evidence for the selection of appropriate target genes and time windows for the regulation of extracellular matrix gene therapy for articular cartilage injury.