[重组热休克蛋白70腺病毒转染在体外保护肠上皮细胞（IEC-6细胞）免受缺氧复氧损伤]

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

作者信息

Li Xiaolu, Yuan Zhiqiang, Peng Yizhi

机构信息

Institute of burn research, Southwest Hospital, The Third Military Medical University, Chongqing 400038, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2002 Aug 25;82(16):1102-4.

PMID:12425818

Abstract

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.

摘要

目的

研究通过重组腺病毒介导热休克蛋白70（HSP70）基因转染来保护肠上皮细胞（IEC-6细胞）免受缺氧复氧损伤的可行性。

方法

用全长人HSP70基因构建重组腺病毒（AdCMVHSP70）。将培养的肠上皮细胞（IEC-6细胞）分为四组：三组分别用AdCMVHSP70转染24小时、48小时和72小时，一组用空重组腺病毒转染（对照组）。转染48小时后通过RT-PCR检测转染的人HSP70基因的过表达情况。四组细胞经历1小时缺氧后再进行1小时复氧，研究不同转染时间（24小时、48小时和72小时）时转染的人HSP70基因过表达的作用。采用MTT法分析细胞活力，体外使用Annexin-V-FLOUS染色试剂盒评估细胞凋亡和死亡细胞比例。

结果

AdCMVHSP70转染的IEC-6细胞中人HSP70基因表达为阳性，对照组为阴性。缺氧复氧后，HSP70基因转染组的细胞活力率分别为87.24±0.60、90.27±0.64和78.52±0.61，均显著高于对照组（均P<0.01），而HSP70基因转染24小时和48小时组的凋亡率分别为3.93±0.28和3.95±0.54，显著低于对照组（均P<0.05），然而，HSP70基因转染72小时组的凋亡率为11.49±1.45，与对照组（11.39±1.62）无显著差异（P>0.05）。HSP70基因转染组的死亡细胞比例分别为8.21±0.26、5.83±0.32和5.83±0.32，显著低于对照组（15.51±0.46，均P<0.01）。保护作用的峰值出现在转染后48小时。