摘要目的 探讨血红素氧合酶-1(HO-1)蛋白转导对大鼠海马神经元氧糖缺失/复氧复糖损伤的影响.方法 采用pET-21a(+)-p53-11R质粒(11R质粒)构建11R-HO-1质粒,鉴定和收集11R-HO-1融合蛋白.采用随机数字表法将培养7d的大鼠海马神经元分为5组(n=171):氧糖缺失/复氧复糖组(OGD/R组)、生理盐水组(NS组)、11R质粒组(11R组)、300 nmol/L 11R-HO-1组(H1组)、1 500nmol/L 11R-HO-1组(H2组).NS组、11R组、H1组和H2组神经元分别经300 nmol/L生理盐水、300nmol/L11R质粒、300 nmol/L11R-HO-1融合蛋白、1 500 nmol/L 11R-HO-1融合蛋白孵育2h后制备氧糖缺失/复氧复糖模型.神经元用5％ CO2-95％N2饱和的无糖/无氧DMEM液和5％ CO2-95％N2培养箱孵育45 min,随后更换回高糖型DMEM培养液放置于37℃、5％CO2-95％空气培养箱培养24h,以制备氧糖缺失/复氧复糖损伤模型.模型制备结束即刻采用MTT法检测神经元存活率,TUNEL法检测神经元凋亡率,Western blot法检测HO-1和caspase-3蛋白的表达水平.结果 与OGD/R组比较,H1组和H2组神经元存活率升高,凋亡率降低,caspase-3蛋白表达下调,HO-1蛋白表达上调(P＜0.05),NS组和11R组上述指标差异无统计学意义(P＞0.05);与H1组比较,H2组神经元存活率升高,凋亡率降低,caspase-3蛋白表达下调,HO-1蛋白表达上调(P＜0.05).结论 HO-1蛋白转导可减轻大鼠海马神经元氧糖缺失/复氧复糖损伤.

abstractsObjective To investigate the effect of transduction of heme oxygenase-1 (HO-1) protein on oxygen-glucose deprivation and restoration (OGD/R)-induced injury to hippocampal neurons in rats.Methods Plasmid 11R-HO-1 was constructed using plasmid pET-21a(+)-p53-11R (plasmid 11R) and 11R-HO-1 fusion protein was identified and collected.Hippocampal neurons obtained from newborn Wistar rats (＜ 48 h) were cultured for 7 days in vitro and then the neurons were randomly divided into 5 groups (n =171 each) using a random number table:OGD/R group,normal saline group (group NS),plasmid 11R group (11R group),300 nmol/L 11R-HO-1 group (H1 group),and 1 500 nmol/L 11R-HO-1 group (H2 group).In NS,11R,H1 and H2 groups,the neurons were incubated for 2 h with 300 nmol/L normal saline,300 nmol/L plasmid 11R,300 nmol/L 11R-HO-1 fusion protein,and 1 500 nmol/L 11R-HO-1 fusion protein,respectively,and then OGD/R was performed.The neurons were incubated in deoxygenated glucose-free DMEM medium and sealed under 5 ％ CO2-95 ％ N2 in an anaerobic chamber equilibrated to 37 ℃ for 45 min.OGD was terminated by replacement of the medium with high glucose DMEM medium and by returning the cultures to a standard incubator maintained at 37 ℃ in 5 ％ CO2-95 ％ air and the neurons were then incubated for 24 h.Immediately after OGD/R was established,the cell survival rate (by MTT assay),apoptosis rate (using TUNEL),and expression of HO-1 and caspase-3 protein (by using Western blot) were measured.Results Compared with group OGD/R,the cell survival rate was significantly increased,the apoptosis rate was decreased,the caspase-3 expression was down-regulated,HO-1 protein expression was up-regulated in H1 and H2 groups (P ＜ 0.05),and no significant change was found in the parameters mentioned above in NS and 11R groups (P ＞ 0.05).Compared with group H1,the cell survival rate was significantly increased,the apoptosis rate was decreased,the caspase-3 expression was down-regulated,and HO-1 protein expression was up-regulated in group H2 (P ＜ 0.05).Conclusion Transduction of HO-1 protein can reduce OGD/R-induced injury to hippocampal neurons of rats.