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.