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目的探讨交联了抗氧化酶的聚合人血红蛋白对过氧化氢(hydrogenperoxide,H2O2)引起的血管内皮细胞氧化损伤的保护作用。方法按照对细胞处理方法的不同,分为正常对照组、单加H2O2组、抗氧化酶交联的多聚血红蛋白(PolyHb-SOD-CAT)+H2O2组、多聚血红蛋白(PolyHb)+H2O2组、单加PolyHb-SOD-CAT组和单加PolyHb组,化学比色法检测细胞内还原型谷胱甘肽(glutathione,GSH)、Ca2+浓度,免疫组化技术检测核转录因子NF-kB在细胞内的分布,RT-PCR技术检测血红素氧化酶(heme oxygenase,HO-1)、内皮型一氧化氮合酶(endothelialnitric oxide synthase,eNOS)的mRNA表达情况,流式细胞术检测细胞死亡情况。结果与对照组相比,单加H2O2组的GSH浓度为(0.006 4±0.003 9)mmol/L,Ca2+浓度为(690.05±145.11)nmol/ml,NF-kB大量位移入核,HO-1、eNOS的mRNA表达量上升,细胞死亡率高;而与单加H2O2组比,PolyHb-SOD-CAT+H2O2组的GSH浓度为(0.0214±0.009 8)mmol/L,Ca2+浓度为(172.43±24.09)nmol/ml,NF-kB绝大部分仍位于胞浆内,HO-1、eNOS的mRNA表达量明显降低,细胞死亡率下降。结论PolyHb-SOD-CAT能够抑制H2O2引起的血管内皮细胞氧化损伤。
Objective To investigate the protective effect of polymerized human hemoglobin with antioxidant enzymes on oxidative damage of vascular endothelial cells induced by hydrogen peroxide (H2O2). Methods According to different treatment methods, cells were divided into normal control group, PolyHb-SOD-CAT + H2O2 group, PolyHb + H2O2 group, PolyHb-SOD-CAT group and PolyHb group alone were added. The levels of intracellular glutathione (GSH) and Ca2 + were measured by chemical colorimetric assay. The expression of NF-kB in nucleus was detected by immunohistochemistry RT-PCR was used to detect the mRNA expression of heme oxygenase (HO-1) and endothelial nitric oxide synthase (eNOS). The cell death was detected by flow cytometry. Results Compared with the control group, GSH concentration in the H2O2 group was (0.006 4 ± 0.003 9) mmol / L, Ca2 + concentration was (690.05 ± 145.11) nmol / ml, NF- The levels of eNOS mRNA and protein in the PolyHb-SOD-CAT + H2O2 group were (0.0214 ± 0.009 8) mmol / L and (172.43 ± 24.09) nmol / ml, the majority of NF-kB is still located in the cytoplasm, HO-1, eNOS mRNA expression was significantly reduced, decreased cell mortality. Conclusion PolyHb-SOD-CAT can inhibit H2O2-induced oxidative damage of vascular endothelial cells.