目的 研究改性柑橘果胶（MCP）对兔关节软骨细胞的糖酵解代谢的影响。 方法 分离、培养兔膝关节软骨细胞至第3代后，分别用含0、500 μg/ml MCP的培养液（MCP0和MCP500）培养3 d，设MCP0为对照组；连续传代培养软骨细胞，每代软骨细胞分别以MCP0和MCP500培养3 d；以白细胞介素-1β（IL-1β）处理软骨细胞1 d后，再分别用MCP0和MCP500培养3 d。以2-脱氧-葡萄糖（2DG）处理软骨细胞1 d后，再分别用MCP0和MCP500培养3 d；于培养3 d后，通过CCK-8方法检测软骨细胞的相对增殖情况；利用葡萄糖和乳酸检测试剂盒测定软骨细胞的葡萄糖摄取量和乳酸生成量；通过免疫荧光染色方法考察连续传代软骨细胞的Ⅱ型胶原α1（COL2A1）合成情况；采用实时荧光定量PCR（RT-qPCR）检测软骨细胞的COL2A1、蛋白聚糖（ACAN）、性别决定区Y框蛋白9（SOX9）、缺氧诱导因子-1α（HIF-1α）、葡萄糖转运蛋白-1（Glut-1）、丙酮酸激酶M2（PKM2）、乳酸脱氢酶A（LDHA）和葡萄糖转运蛋白-3（Glut-3）的基因表达情况。 结果 与对照组相比，MCP处理提高了软骨细胞的葡萄糖摄取量和乳酸生成量，上调了ACAN、HIF-1α、Glut-1和PKM2的基因表达水平；与对照组相比，连续传代过程中，MCP可以促进软骨细胞的增殖、维持软骨细胞的表型，上调COL2A1、ACAN、SOX9、HIF-1α、Glut-1、PKM2和LDHA的基因表达，提高软骨细胞COL2A1合成量和乳酸生成量；在经IL-β处理后，与对照组相比，MCP处理提高了软骨细胞的葡萄糖摄取量，上调了COL2A1、ACAN、HIF-1α和Glut-1的基因表达。在经2DG处理后，MCP处理提高了软骨细胞的葡萄糖摄取量，上调SOX9、HIF-1α、PKM2和Glut-3的基因表达。 结论 MCP能够增强软骨细胞的葡萄糖摄取能力，提高软骨细胞糖酵解代谢水平。 Objective To investigate the effect of modified citrus pectin (MCP) on the glucose metabolism of rabbit articular chondrocytes. Methods The third generation (P3) rabbit knee chondrocytes were extracted and cultured with 0 μg/ml (MCP0, control group) and 500 μg/ml of MCP (MCP500) for 3 days. Chondrocytes (P2-P7)were cultured continuously, and each generation of chondrocytes was treated with MCP0 and MCP500 medium for 3 days. Chondrocytes were treated with interleukin-1β (IL-1β) for 1 day and then treated with MCP0 and MCP500 medium for 3 days, respectively. Chondrocytes were treated with 2-deoxy-glucose (2DG) for 1 day and then treated with MCP0 and MCP500 medium for 3 days, respectively. After three days of culture, the proliferation of chondrocytes was calculated by CCK-8. Glucose uptake activity and lactate production of chondrocytes were measured by glucose and lactate detection kits. The synthesis of type Ⅱ collagen (COL2A1) in sequential chondrocytes was investigated by immunofluorescence staining. The gene expression of COL2A1, proteoglycan (ACAN), SOX9, hypoxia-inducible factor-1α (HIF-1α), glucose transporter-1 (Glut-1), pyruvate kinase M2 (PKM2), lactate dehydrogenase-A (LDHA) and glucose transporter-1 (Glut-3) were further detected by RT-qPCR. Results Compared with the control group, MCP treatment could increase the glucose uptake activity and lactate production of chondrocytes, and enhance the gene expression ability of HIF-1α, Glut-1, PKM2 and ACAN. Besides, MCP treatment could stimulate chondrocyte proliferation, maintain chondrocyte phenotype, increase lactate production, and upregulate the expression of COL2A1, ACAN, SOX9, HIF-1α, Glut-1, PKM2 and LDHA. After the treatment with IL-1β, MCP treatment could increase glucose uptake activity and upregulate the expression ofCOL2A1, ACAN, HIF-1α and Glut-1. After treatment with 2DG, MCP treatment could increase glucose uptake activity and upregulate the expression of SOX9, HIF-1α, PKM2 and Glut-3 genes. Conclusions MCP can enhance the glucose uptake capacity of chondrocytes and increase the level of chondrocyte glycolytic metabolism.
Effect of modified citrus pectin on glycolysis of rabbit articular chondrocytes
Objective To investigate the effect of modified citrus pectin (MCP) on the glucose metabolism of rabbit articular chondrocytes. Methods The third generation (P3) rabbit knee chondrocytes were extracted and cultured with 0 μg/ml (MCP0, control group) and 500 μg/ml of MCP (MCP500) for 3 days. Chondrocytes (P2-P7)were cultured continuously, and each generation of chondrocytes was treated with MCP0 and MCP500 medium for 3 days. Chondrocytes were treated with interleukin-1β (IL-1β) for 1 day and then treated with MCP0 and MCP500 medium for 3 days, respectively. Chondrocytes were treated with 2-deoxy-glucose (2DG) for 1 day and then treated with MCP0 and MCP500 medium for 3 days, respectively. After three days of culture, the proliferation of chondrocytes was calculated by CCK-8. Glucose uptake activity and lactate production of chondrocytes were measured by glucose and lactate detection kits. The synthesis of type Ⅱ collagen (COL2A1) in sequential chondrocytes was investigated by immunofluorescence staining. The gene expression of COL2A1, proteoglycan (ACAN), SOX9, hypoxia-inducible factor-1α (HIF-1α), glucose transporter-1 (Glut-1), pyruvate kinase M2 (PKM2), lactate dehydrogenase-A (LDHA) and glucose transporter-1 (Glut-3) were further detected by RT-qPCR. Results Compared with the control group, MCP treatment could increase the glucose uptake activity and lactate production of chondrocytes, and enhance the gene expression ability of HIF-1α, Glut-1, PKM2 and ACAN. Besides, MCP treatment could stimulate chondrocyte proliferation, maintain chondrocyte phenotype, increase lactate production, and upregulate the expression of COL2A1, ACAN, SOX9, HIF-1α, Glut-1, PKM2 and LDHA. After the treatment with IL-1β, MCP treatment could increase glucose uptake activity and upregulate the expression ofCOL2A1, ACAN, HIF-1α and Glut-1. After treatment with 2DG, MCP treatment could increase glucose uptake activity and upregulate the expression of SOX9, HIF-1α, PKM2 and Glut-3 genes. Conclusions MCP can enhance the glucose uptake capacity of chondrocytes and increase the level of chondrocyte glycolytic metabolism.
万方数据
