腺病毒介导的热休克蛋白70在神经元和胶质细胞中的抗缺氧研究  

Recombinant heat shock protein 70 induced by adenovirus protects neurons and glial cells against hypoxia/reoxygenation injury

作　　者：杨芳芳[1] 徐小娜 胡丹[1] 时飞[3] 曲彦[1] 

机构地区：[1]青岛大学医学院附属市立医院ICU,山东青岛,266011 [2]青岛市卫生学校 [3]青岛大学医学院附属市立医院麻醉科,山东青岛,266011

出　　处：《中华危重病急救医学》2008年第11期-,共5页Chinese Critical Care Medicine

基　　金：山东省自然科学基金，山东省青岛市市南区科技局资助项目

摘　　要：目的 探讨重组腺病毒介导的热休克蛋白70(HSP70)表达对神经元和胶质细胞缺氧/再复氧损伤的保护作用.方法 制备携带全长HSP70基因的重组腺病毒vAd-HSP70.感染体外培养的神经元和胶质细胞,检测靶细胞中外源性HSP70的表达.感染vAd-HSP70 24、48、72 h组和感染vAd-GFP对照组的细胞经缺氧/再复氧处理后,分别测定细胞活性、细胞培养上清液中乳酸脱氢酶(LDH)活性及线粒体和胞质内细胞色素C含量.结果 感染vAd-HSP70的神经细胞可检测到外源性HSP70基因表达.经缺氧/再复氧处理,感染vAd-HSP70组细胞活性较感染vAd-GFP对照组明显增强(P均＜0.05);感染vAd-HSP70 24、48、72 h组细胞培养上清液中LDH活性[(1 480±121)、(1 023士106)、(1 132±197)U/L]均明显低于感染vAd-GFP对照组[(1 976±190)U/L],线粒体中细胞色素C含量(0.986±0.012、1.028±0.007、1.014±0.008)均明显高于感染vAd-GFP对照组(0.970±0.003),而胞质中的细胞色素C含量(0.987±0.008、0.960±0.005、0.964±0.003)则低于感染vAd-GFP对照组(1.011±0.005,P＜0.05或P＜0.01),其中以感染48 h最为理想(P均＜0.01).结论 腺病毒介导的外源性HSP70表达可保护神经元和胶质细胞抵抗缺氧/再复氧损伤,具有明确的细胞保护作用.Objective To investigate the protective effect on neurons and glial cells against hypoxia/reoxygenation injury with recombinant heat shock protein 70 (HSP70) induced by adenovirus.Methods Neurons and glial cells in euhure were divided into four groups : three groups were treated with recombinant adenovirus (vAd-HSP70) transfected human HSP70 gene at 24,48 and 72 hours respectively,and vAd-GFP transfected cell served as control.Cells in different groups were subjected to hypoxia/reoxygenation,then the cell viability was analyzed by methyl thiazolyl tetrazolium (MTT) method,lactate dehydrogenase (LDH) viability was evaluated with LDH staining kit,and cytoehrome C (Cyt C) in mitochondria and cytoplasm were assessed by Western blotting. Results The expression of human HSP70 gene was detected in the vAd-HSP70 transfeetion group.After hypoxia/reoxygenation treatment,the cell viability in transfected groups was higher than that of control group (all P＜0.05),the LDH viability of vAd-HSPT0 transfected groups at different time points was 1 480±121,1 023±106,and (1 132±197) U/L respectively,and they were significantly lower than control group[(1 976± 190) U/L,all P＜0.01].In transfected groups,the content of Cyt C in mitochondria (0.986±0.012,1.028± 0.007,1.014±0.008) was significantly higher than control group (0.970±0.003,P＜0.05 or P＜0.01).In contrast,the content of Cyt C in cytoplasm (0.987±0.008,0.960±0.005,0.964±0.003) was lower than that of control group (1.011±0.005,all P＜0.01).The protective effect was especially obvious when the cells were transfected by vAd-HSP70 at 48 hours (all P＜0.01).Conclusion The expression of human HSP70 mediated by recombinant adenovirus may protect neurons and glial cells against hypoxia/reoxygenation in vitro.

关 键 词：热休克蛋白70 腺病毒载体 神经细胞 缺氧再复氧 

分 类 号：R686[医药卫生—骨科学]