Yang Fang-fang, Xu Xiao-na, Hu Dan, Shi Fei, Qu Yan
Intensive Care Unit, The Affiliated Qingdao Municipal Hospital of Qingdao University Medical College, Qingdao 266011, Shandong, China.
Zhongguo Wei Zhong Bing Ji Jiu Yi Xue. 2008 Nov;20(11):681-4.
To investigate the protective effect on neurons and glial cells against hypoxia/ reoxygenation injury with recombinant heat shock protein 70 (HSP70) induced by adenovirus.
Neurons and glial cells in culture 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 cytochrome C (Cyt C) in mitochondria and cytoplasm were assessed by Western blotting.
The expression of human HSP70 gene was detected in the vAd-HSP70 transfection 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-HSP70 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).
The expression of human HSP70 mediated by recombinant adenovirus may protect neurons and glial cells against hypoxia/reoxygenation in vitro.
探讨腺病毒介导的重组热休克蛋白70（HSP70）对神经元和神经胶质细胞缺氧/复氧损伤的保护作用。
将培养的神经元和神经胶质细胞分为四组：三组分别在24、48和72小时用转染人HSP70基因的重组腺病毒（vAd-HSP70）处理，以转染vAd-GFP的细胞作为对照。对不同组的细胞进行缺氧/复氧处理，然后用噻唑蓝（MTT）法分析细胞活力，用乳酸脱氢酶（LDH）染色试剂盒评估LDH活力，并用蛋白质免疫印迹法检测线粒体和细胞质中的细胞色素C（Cyt C）。
在vAd-HSP70转染组中检测到了人HSP70基因的表达。缺氧/复氧处理后，转染组的细胞活力高于对照组（均P<0.05），vAd-HSP70转染组在不同时间点的LDH活力分别为1480±121、1023±106和（1132±197）U/L，均显著低于对照组[（1976±190）U/L，均P<0.01]。在转染组中，线粒体中Cyt C的含量（0.986±0.012、1.028±0.007、1.014±0.008）显著高于对照组（0.970±0.003，P<0.05或P<0.01）。相反，细胞质中Cyt C的含量（0.987±0.008、0.960±0.005、0.964±0.003）低于对照组（1.011±0.005，均P<0.01）。当细胞在48小时用vAd-HSP70转染时，保护作用尤为明显（均P<0.01）。
重组腺病毒介导的人HSP70表达可在体外保护神经元和神经胶质细胞免受缺氧/复氧损伤。
