氧化应激对人成骨样细胞MG63活性的影响

Effect of oxidative stress on the activity of human osteoblastic MG63 cells

目的建立人成骨样细胞氧化应激模型,研究氧化应激对人成骨样细胞形态和增殖活力的影响。方法用不同浓度黄嘌呤/黄嘌呤氧化酶(xanthine/xanthine oxidase,X/XO)的酶促反应产生的超氧阴离子自由基(O_2^-˙)刺激人成骨样细胞MG63,建立成骨细胞内氧化应激模型,用X/XO加黄嘌呤氧化酶抑制剂羟嘌呤醇研究其对该氧化应激模型的逆转作用。运用氧化敏感性荧光探针2'7'-二氯荧光黄双乙酸盐结合流式细胞术检测细胞内活性氧(reactive oxygen species,ROS)的生成,用倒置相差显微镜和MTT实验观察研究氧化应激对成骨细胞形态及增殖活力的影响。结果 X/XO处理MG63细胞后,细胞形态发生明显破坏;在相同处理时间下,X/XO浓度越高,细胞内ROS荧光强度值越高,吸光光密度(opitical density,OD)值越低,差异具有统计学意义(P<0.05),且X/XO加羟嘌呤醇联合处理组的细胞内ROS平均荧光强度较相同浓度X/XO单独处理组明显降低(P<0.05)。在相同X/XO处理浓度下,随着处理时间的延长,细胞内ROS平均荧光强度逐渐增强,OD值明显降低,120 min时细胞内ROS平均荧光强度比对照组增加了345%。24 h时OD值为相同时间对照组的22.9%。结论 X/XO可导致人成骨样细胞氧化应激损伤,破坏人成骨样细胞的形态,抑制人成骨样细胞的增殖活力。X/XO抑制剂羟嘌呤醇可逆转X/XO引起的人成骨样细胞氧化损伤。

Objective To investigate the morphology and proliferation viability in oxidative stress induced damage in human MG63 cells. Methods The MG63 cells were treated with superoxide anion(O_2^-.) produced by different concentrations of xanthine/xanthine oxidase enzymatic reactions to establish the model of oxidative stress in MG63 cells, using the xanthine oxidase inhibitor oxypurinol to observe the reverse effect of oxypurinol on xanthine/xanthine oxidase induced damage in human MG63 cells. Using the flow cytometry, the production of intracellular reactive oxygen species(ROS) induced by xanthine/xanthine oxidase induced cellular oxidative stress damage was evaluated by the oxidationsensitive fluorescent probe, the 2'7'-dichlorofluorescin diacetate. Cellular viability and morphology was evaluated by the MTT assay and the phase contrast microscope. Results Xanthine/xanthine oxidase induced intracellular ROS production in a dose-and time-dependent manner(P < 0.05). The cellular viability was reduced and cellular morphology was damaged, too(P < 0.05). Xanthine/xanthine oxidase induced the damage of the cellular morphology. At the same processing time, the higher the xanthine/xanthine oxidase concentration, the higher intracellular ROS fluorescence intensity value, and the lower OD value, the difference was statistically significant(P < 0.05). The intracellular mean ROS fluorescence intensity in xanthine/xanthine oxidase + oxypurinol combined treatment group was significantly lower compared with the same concentration of xanthine/xanthine oxidase(P < 0.05). At the same concentration of xanthine/xanthine oxidase, with the extension of treatment time, the intracellular mean ROS fluorescence intensity gradually increased, the OD value decreased, compared with the control group, the intracellular mean ROS fluorescence intensity of120 min increased to 345% of the control, was the highest among the xanthine/xanthine oxidase groups. The OD value of 24 h was the 22.9% of the control group, was the lowest among the xanthine/xanthine oxidase groups, cell proliferation activity decreased more obvious. Conclusions Xanthine/xanthine oxidase could induce oxidative stress damaged the cellular morphology and reduced the cellular viability in MG63 cell lines. The oxypurinol(the inhibitor of xanthine oxidas) could reverse the oxidative stress injury induced by xanthine/xanthine oxidase in human osteoblastic cells.