荷载HO-1基因的脑靶向纳米粒对缺血性脑病大鼠海马神经元的保护作用

摘要

目的：探讨荷载血红素加氧酶-1（ HO-1）基因的乳铁蛋白与聚乙二醇修饰的阳离子壳聚糖（ Lf-PEG-QMC）纳米粒对缺血性脑病大鼠海马神经元的保护作用。方法将24只SD大鼠随机分为假手术组、缺血性脑病组、缺血性脑病＋HO-1基因组（纯基因组）、缺血性脑病＋荷载HO-1基因的Lf-PEG-QMC纳米粒干预组（纳米粒组），每组各6只。缺血性脑病组、纯基因组、纳米粒组采用四动脉阻塞法制备缺血性脑病模型，假手术组仅分离而不结扎双侧颈总动脉。缺血性脑病组于缺血15 min后松开血管夹进行再灌注；纯基因组缺血15 min后再灌注时由尾静脉注射HO-1基因质粒20μg/kg；纳米粒组缺血15 min后再灌注时由尾静脉注射荷载HO-1基因的Lf-PEG-QMC纳米粒（按基因剂量为20μg/kg给药）；假手术组不行再灌注操作。再灌注后第5天将各组大鼠麻醉后取脑组织制作切片，采用HE染色法在倒置显微镜下（×400）检测海马CA1区神经元存活数量，计算神经元存活率。结果假手术组、缺血性脑病组、纯基因组、纳米粒组神经元存活率分别为100％、11．0％、16．6％、57．6％，缺血性脑病组、纯基因组与纳米粒组神经元存活率均低于假手术组（P＜0．05），纳米粒组神经元存活率高于缺血性脑病组及纯基因组（ P＜0．05），纯基因组与缺血性脑病组相比无统计学差异。结论 Lf-PEG-QMC纳米粒能有效携带HO-1基因跨越BBB并在脑组织中表达，有助于HO-1发挥对缺血性脑病海马神经元的保护作用。%Objective To investigate the protective efficacy of HO-1 plasmid loaded Lf-PEG-QMC nanoparticles on the hippocampal neurons of rats with cerebral ischemic injury.Methods A total of 24 adult SD rats were randomly divided into 4 groups: pseudo-operating group, ischemia-reperfusion injury group, ischemia-reperfusion injury +HO-1 plasmid group ( plasmid group ) , andischemia-reperfusion injury +HO-1 plasmid loaded Lf-PEG-QMC nanoparticles group ( NPs group) .Models of cerebral ischemia were established by the method of four vessels occlusion in ischemia-reperfusion injury group, plasmid group and NPs group.After the common carotid arteries were occluded for 15 minutes, reperfusion was con-ducted by vein in each group as 20 μg/kg HO-1 plasmids administered in plasmid group, HO-1 plasmid loaded Lf-PEG-QMC nanoparticles administered in NPs group.Rats in pseudo-operating group did not adopted the reperfusion.After 5 days of ischemia-reperfusion, rats brain in each group were extracted to make histologic sections.The survival statuses of neurons in hippocampal CA1 sector of each rats were detected by HE staining method and to calculate the survival neurons rate.Results The survival neurons rate of pseudo-operating group, ischemia-reperfusion injury group, plasmid group and NPs group was 100%, 11.0%, 16.6%, 57.6%, respectively.The survival neurons rate of ischemia-reperfusion injury group, plasmid group and NPs group was lower than that of pseudo-operating group(P<0.05).The survival neurons rate of NPs group was higher than that of ischemia-reperfusion injury group and plasmid group(P<0.05).There was no statis-tic difference between ischemia-reperfusion injury group and plasmid group.Conclusions Lf-PEG-QMC nanoparticles was effective in the transfer of HO-1 plasmid across BBB and it's stable express in brain, which indicates that Lf-PEG-QMC nanoparticles was helpful in the effect of HO-1 on neurons in cerebral ischemic injury.