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目的研究后交叉韧带(posterior cruciate ligament,PCL)成纤维细胞与滑膜细胞共培养下,2种细胞之间的交流对PCL成纤维细胞中基质金属蛋白酶MMP-1、2、3表达及活性的影响。方法分为共培养组和单培养组:①用显微镜观察共培养24 h后细胞的活性和迁移率。②分别培养6 h后,提取总RNA,逆转录PCR;实时荧光定量PCR对人后交叉韧带成纤维细胞MMP-1、2、3基因的表达进行半定量和定量分析。③共培养处理1、2、3 d后收集培养上清液,用明胶酶谱法检测MMP-2的活性。结果共培养使PCL细胞和滑膜细胞较单层细胞培养迁移率分别增高了(43.1±0.1)%和(76.2±0.2)%(P<0.01)。与单培养相比,共培养组明显增加了MMP-2的基因表达,但降低了MMP-1、3的基因表达。酶谱结果表明共培养增加了MMP-2活性(P<0.05)。结论共培养能够促进细胞的迁移以及调节细胞中MMP-1、2、3的表达情况。
Objective To study the expression and activity of MMP-1, MMP-2 and MMP-2 in PCL fibroblasts after co-culture of posterior cruciate ligament (PCL) fibroblasts and synovial cells with two kinds of cells influences. The method was divided into co-culture group and single culture group: ① The activity and migration rate of cells after 24 h co-culture were observed with microscope. ② After cultured for 6 h, total RNA was extracted and reverse-transcriptase PCR was performed. Semi-quantitative and quantitative analysis of MMP-1 and MMP-2 and MMP-2 were performed by real-time fluorescence quantitative PCR. ③ After co-culture for 1, 2 and 3 days, the supernatant was collected and the activity of MMP-2 was detected by gelatin zymography. Results Co-culture increased (43.1 ± 0.1)% and (76.2 ± 0.2)% (P <0.01) in PCL cells and synovial cells, respectively. Compared with the single culture, co-culture group significantly increased the gene expression of MMP-2, but reduced the gene expression of MMP-1 and 3. Zymogram results showed that co-culture increased MMP-2 activity (P <0.05). Conclusion Co-culture can promote the migration of cells and regulate the expression of MMP-1,2,3 in cells.