重组腺病毒介导的HSP70转染对肠上皮细胞缺氧再复氧损伤的防护作用

目的　研究重组腺病毒介导的热休克蛋白 70转染对肠上皮细胞缺氧再复氧损伤的保护作用。方法　将重组含人全长HSP70基因的腺病毒载体 (AdCMVHSP70 )转染体外培养的肠上皮细胞株IEC 6 ,检测转染细胞HSP70的基因表达水平。对照组 (转染空载体组 )、转染HSP70 2 4h、48h及72h组IEC 6细胞经缺氧再复氧处理后 ,分别对细胞的活力、凋亡及死亡水平进行检测分析。结果AdCMVHSP 70转染组细胞HSP70基因表达为阳性 ,对照组无表达。经缺氧再复氧处理后 ,AdCMVHSP70转染组细胞活力较对照组明显增强 (P <0 0 1 ) ,Annexin V Flous试剂盒检测死亡细胞明显减少 (P<0 0 1 ) ,凋亡有一定水平的抑制 (P <0 0 5)。结论　重组腺病毒介导的HSP70转染可保护肠上皮细胞抵抗缺氧再复氧损伤 ,具有明确的细胞保护作用。而其具体保护机制可能与抑制细胞凋亡有关

Recombinant heat shock protein 70 adenovirus transfection protects intestinal epithelial cells (IEC-6 cells) against hypoxia-reoxygenation in vitro

OBJECTIVE: To study the feasibility of protection of intestinal epithelial cells (IEC-6 Cells) against hypoxia-reoxygenation through recombinant adenovirus-mediated heat shock protein 70 (HSP70) gene transfection. METHODS: Recombinant adenovirus (AdCMVHSP70) was constructed with full length human HSP70 gene. Cultured intestinal epithelial cells (IEC-6 cells) were divided into four groups: three groups transfected with AdCMVHSP70 for 24 h, 48 h and 72 h respectively, and one group transfected with vacant recombinant adenovirus (control group). The over expression of transfected human HSP70 gene was detected by RT-PCR 48 h after transfection. After the cells in the four groups suffered 1 hour of hypoxia followed by 1 hour of reoxygenation, the effects of transfected human HSP70 gene overexpression during different transfection times (24 h, 48 h, and 72 h) were studied. The cell viability was analyzed by MTT method, and the apoptosis and dead cell ratio were evaluated with Annexin-V-FLOUS staining kit in vitro. RESULTS: The expression of human HSP70 gene was positive in the AdCMVHSP70 transfected IEC-6 cells and negative in the control group. After hypoxia-reoxygenation, the cell viability rates of HSP70 gene transfected groups were 87.24 +/- 0.60, 90.27 +/- 0.64, and 78.52 +/- 0.61 respectively, all significantly higher than that of control group (all P < 0.01), while the apoptosis rates of HSP70 gene transfected 24 h and 48 h groups were 3.93 +/- 0.28 and 3.95 +/- 0.54 respectively, significantly lower than that of control group (both P < 0.05), however, the apoptosis rate of HSP70 gene transfected 72h group was 11.49 +/- 1.45, not significantly different from that of control group (11.39 +/- 1.62, P > 0.05). The dead cell ratios of HSP70 gene transfected groups were 8.21 +/- 0.26, 5.83 +/- 0.32, and 5.83 +/- 0.32 respectively, significantly lower than that of control group (15.51 +/- 0.46, all P < 0.01). The peak of protection was at 48 h after the transfection. CONCLUSION: The over expression of human HSP70 mediated by recombinant adenovirus protects intestinal epithelial cells (IEC-6 cells) against hypoxia-reoxygenation in vitro. The probable mechanism may be concerned with the inhibition of cell apoptosis after hypoxia-reoxygenation.