糖皮质激素对骨髓微血管内皮细胞活性氧代谢影响的实验研究

EFFECT OF GLUCOCORTICOID ON PRODUCTION OF REACTIVE OXYGEN SPECIES IN BONE MICROVASCULAR ENDOTHELIAL CELLS

目的糖皮质激素的应用是引起非创伤性股骨头缺血性坏死的主要原因之一。通过研究经糖皮质激素处理后的骨髓微血管内皮细胞活性氧(reactive oxygen species,ROS)的代谢变化,进一步探讨激素性股骨头缺血性坏死的发病机制。方法取行人工全髋关节置换术患者自愿捐献的股骨头内松质骨,利用酶消化法分离培养骨髓微血管内皮细胞,倒置相差显微镜下观察细胞形态。取第3代细胞,分别与不同浓度(0、0.03、0.10、0.30、1.00 mg/mL)氢化可的松培养后,采用MTT法测定细胞增殖抑制率;流式细胞仪观测细胞凋亡率;采用荧光探针检测细胞中ROS含量变化,以及与ROS代谢相关的黄嘌呤氧化酶(xanthine oxidase,XOD)含量变化。结果原代培养2～3 d后,镜下见单个细胞为梭形,呈铺路石样排列;1周后细胞趋于融合状态。0.03、0.10、0.30、1.00 mg/mL组细胞增殖抑制率分别为20.22%±2.97%、22.94%±4.52%、43.98%±3.35%、78.29%±3.85%,0.30、1.00 mg/mL组的细胞增殖抑制率明显高于0.03、0.10 mg/mL组(P<0.05)。0、0.03、0.10、0.30、1.00 mg/mL组细胞凋亡率分别为0.10%±0.01%、0.23%±0.02%、1.83%±0.04%、6.34%±0.11%、15.33%±0.53%,0.30、1.00 mg/mL组细胞凋亡率明显高于0 mg/mL组(P<0.05)。0、0.30、1.00 mg/mL组ROS含量分别为57.35±7.11、120.47±15.68、166.15±11.57,XOD含量分别为0.017 9±0.000 9、0.028 3±0.001 7、0.067 7±0.004 1;以上两指标3组间两两比较差异均有统计学意义(P<0.05)。结论糖皮质激素通过使血管内皮细胞内ROS生成增加,引起氧化应激反应,进而导致细胞功能受损。

Objective Glucocorticoid is the main cause of non-traumatic avascular necrosis of femoral head.To explore the changes of reactive oxygen species（ROS） in the bone microvascular endothelial cells treated with glucocorticoid so as to investigate the pathogenesis of steroid-induced avascular necrosis of femoral head.Methods The cancellous bone of femoral head was harvested from voluntary donators undergoing total hip arthroplasty,and then the bone microvascular endothelial cells were isolated by enzyme digestion.The cells at passage 3 were cocultured with different concentrations of hydrocortisone（0,0.03,0.10,0.30,and 1.00 mg/mL） for 24 hours.MTT assay was used for the inhibitory rate of cell proliferation,flow cytometry for apoptosis rate,and fluorescence probe for the production of ROS and xanthine oxidase（XOD）.Results At 2-3 days primary culture,the cells were spindle and arranged like cobbles and they reached confluence after 1 week.The inhibitory rates of cell proliferation in 0.03,0.10,0.30,and 1.00 mg/mL groups were 20.22%±2.97%,22.94%±4.52%,43.98%±3.35%,and 78.29%±3.85%,respectively;and 2 high-concentration groups（0.30 and 1.00 mg/mL groups） were significantly higher（P0.05） than 2 low-concentration groups（0.03 and 0.10 mg/mL groups）.The apoptosis rates in 0,0.03,0.10,0.30,and 1.00 mg/mL groups were 0.10%±0.01%,0.23%±0.02%,1.83%±0.04%,6.34%±0.11%,and 15.33%±0.53%,respectively;2 high-concentration groups（0.30 and 1.00 mg/mL groups） were significantly higher（P0.05） than 0 mg/mL group.In 0,0.30,and 1.00 mg/ mL groups,the ROS levels were 57.35±7.11,120.47±15.68,and 166.15±11.57,respectively,and the XOD levels were 0.017 9±0.000 9,0.028 3±0.001 7,and 0.067 7±0.004 1,respectively;there were significant differences in the levels of ROS and XOD among 3 groups（P0.05）.Conclusion Increasing of ROS production in bone microvascular endothelial cells can be induced by high concentration glucocorticoid,and it can result in cell injury.