Recombinant Heat Shock Protein 70(HSP70) Adenovirus Infection Protects Neurons And Glial Cells Against Hypoxia-reoxygenation

Objective To investigate the effect of heat shock protein 70(HSP70) expressions in neuronal cells with different hypoxia time,and to observe the protective effect of neurons and glial cells against hypoxia-reoygenation through recombinant adenovirus-mediated expression of heat shock protein 70(HSP70) gene.Methods①Cultured neuronal cells with different hypoxia time were divided six groups(hypoxia Oh,0.5h,1h,2h,3h and 4h) were infected with recombinant adenovirus(vAd-HSP70) at 48h.Cells in different groups were treated by hypoxia-reoxygenation,then the expression of HSP70 gene was examined by RT-PCR and Western blotting.②The target cells were divided into four groups:three groups were infected with recombinant adenovirus(vAd-HSP70) carrying human HSP70 gene at 24h,48h and 72h respectively,and vAd-GFP infected cell was as control. Cells in different groups were treated by hypoxia-reoxygenation,then the cell viability was analyzed by MTT method,LDH viability was evaluated with LDH staining kit,and Cyt.C in mitochondria and cytoplasm were tested by Western blotting.Results①The expression of human HSP70 gene was tested in the vAd-HSP70 infected group.②After hypoxia-reoxygenation treatment,expression of HSP70 mRNA and protein were at higher level within hypoxia 4h,reached its peak at hypoxia 1h,and next was at hypoxia 2h.At hypoxia 4h group was lowest than other groups,and there was statistics significance compared with other groups(P＜0.05).③The cell viability in infected groups was higher than that of control group(P＜0.01),the LDH viability of vAd-HSP70 infected groups at different time were 1480±121,1023±106,1132±197 respectively,which significantly lower than control group(1976±190,P＜0.01).In infected groups,the content of Cyt.C in mitochondria (0.9856±0.0121,1.0277±0.0073,1.0136±0.0078) were higher than control group (0.9695±0.0031,P＜0.05).Inversely,the content of Cyt.C in cytoplasm (0.9868±0.0076,0.9600±0.0049,0.9645±0.0028)was lower than that of control group(1.0109±0.0049,P＜0.01).And the protective effect was especially obvious when the cells were infected by vAd-HSP70 at 48h.Conclusions①The expression of HSP70 was at high level when the cultured neuronal cells were treated reoxygenation within hypoxia 2h and may also promote the functional recovery.②The expression of human HSP70 mediated by recombinant adenovirus may protect neurons and glial cells against hypoxia-reoxygenation in vitro.