低氧环境对小鼠未成熟关节透明软骨细胞培养的影响

The effects of hypoxia on immature murine articular chondrocyte in vitro

目的研究低氧和低氧模拟化合物氯化钴对小鼠未成熟关节透明软骨细胞氧感应基因和细胞表型的影响。方法经酶消化分离出小鼠未成熟关节透明软骨细胞，分别在21％氧、2％氧和150μmol/L氯化钴条件下培养一定时间。通过倒置显微镜、透射电镜和扫描电镜观察细胞形态学变化。应用定量PCR和Western Blot检测葡萄糖转运体-1（GLUT-1）、葡萄糖转运体-3（GLUT-3）、磷酸果糖激酶-1（PGK-1）和低氧诱导因子-1α（HIF—1α）的表达。应用定量PCR观察软骨细胞表型改变。应用四甲基偶氮唑盐法（MTT）检测低氧及氯化钴对软骨细胞活性的影响。用葡萄糖检测试剂盒测葡萄糖摄取量。结果不同培养条件下软骨细胞形态无明显差异。2％氧和氯化钴可增加GLUT-1、GLUT-3及PGK-1mRNA表达。2％氧和氯化钴可促进GLUT-1、GLUT-3和HIF—1α蛋白表达。低氧和氯化钴促进细胞活性，增加葡萄糖摄取并促进细胞外基质合成。结论软骨细胞能通过调节氧感应基因适应低氧环境，HIF—1α可能起关键作用。低氧能在一定程度上增加软骨细胞活性和细胞外基质合成。模拟体内氧环境培养细胞能更好地了解软骨细胞特性。

Objective The goal of this study was to determine the effects of hypoxia （2%） and CoCl2 on oxygen-regulated genes and phenotype of immature murine articular chondrocytes in vitro. Methods Enzymatically isolated immature murine articular chondrocytes were cultured for different periods in 21%O2, 2%O2, or 150 μmol/L CoCl2. The morphologic changes of chondrocytes were investigated by inversion microscope and electron microscope. The expression of GLUT-1, GLUT-3, PGK-1, and HIF-1α was determined by real-time PCR and Western Blot. The cell viability was determined by MTT. The changes of chondrocyte phenotype were also quantified by PCR. Glucose uptake in the culture supernatants was measured using the glucose assay kit. Results The shape of chondrocytes didn＇t change in different conditions. Hypoxia and CoCl2 increased the expression of GLUT-1, GLUT-3, PGK-1 and HIF-1α. They also promoted the cell viability and glucose uptake. The increased extrocellular matrixes were found due to stimulation of hypoxia and CoCl2. Conclusion The results suggest that the chondrocytes have the capacity to detect and respond to hypoxia with changes in expression of oxygen-regulated genes. HIF-1α may play a key role in the process. Hypoxia promotes chondrocytic viability and cartilage matrix synthesis. In hypoxic conditions, which mimic the in vivo condition of cartilage, the characteristics of chondrocytes are better understood.