软骨藻酸对人神经胶质瘤细胞的氧化损害及血红素氧化酶-1表达的诱导

Effects on Oxidative Damage and Heme Oxygenase-1Protein Expression Induced by Domoic Acid in H4Human Neuroglioma Cells

目的探讨软骨藻酸(domoicacid)对人神经胶质瘤(H4)细胞的氧化损害及对血红素氧化酶-1(hemeoxygenase-1,HO-1)蛋白的诱导。方法对体外培养的H4细胞分别给予0.064、0.64、6.4μmol/L软骨藻酸,24h后测定各剂量组与对照组细胞生存率、丙二醛(MDA)含量,并采用免疫组化和流式细胞术检测HO-1蛋白表达。结果在四甲基偶氮唑盐微量酶反应比色法(MTT)实验中H4细胞生存率随软骨藻酸剂量增大而下降,呈明显的剂量依赖关系。各剂量组MDA含量与对照组差异均有显著性(P<0.01),6.4μmol/L组MDA含量与0.064μmol/L组差异也有显著性(P<0.01)。免疫组化显示,对照组细胞胞浆HO-1弱阳性染色,0.064、0.64、6.4μmol/L组分别呈低、中、高密度阳性染色。流式细胞术显示各剂量组HO-1荧光强度与对照组差异均有显著性(P<0.01),6.4μmol/L组与0.064μmol/L组差异也有显著性(P<0.01)。结论软骨藻酸对H4细胞具有细胞毒作用和氧化损害作用,并可能通过氧化损害间接诱导HO-1蛋白表达增多。

Objective To study the effects on oxidative damage and heme oxygenase-1(HO-1)protein expression in H4human neuroglioma cells induced by domoic acid.Methods In this study,MTT test,immunity histochemistry stain-ing method and flow cytometry were applied to assess the survival rate,content of MDA and HO-1protein expression level of cultured H4human neuroglioma cells at24h co-cultivation by0.064μmol/L,0.64μmol/L,and6.4μmol/L do-moic acid in vitro.Results The results of MTT test showed that the survival rate of cultured H4human neuroglioma cells was decreased with the increasing of dosage of the domoic acid and showed a dose-depended relationship.The MDA content in each exposed group was significantly different from the control group(P<0.01)and in6.4μmol/L dose group was significantly different from that in0.064μmol/L dose group(P<0.01).The HO-1expression level could be weakly detected in the control group,moderately detected in0.64μmol/L dose group,and strongly detected in6.4μmol/L dose group determined using immunity histochemistry staining method.The result obtained by flow cytometry showed that the fluorescence intensity of HO-1in each dose group was significantly different from that in the control group(P<0.01)and in6.4μmol/L dose group was significantly different from that of0.064μmol/L dose group(P<0.01).Conclusion The results suggested that domoic acid could affect cells survival rate and induce oxidative damage,which might lead to enhancement of HO-1protein expression.