摘要
目的了解Ad-热休克蛋白70（HSP70）对缺氧／复氧损伤后肠上皮细胞线粒体功能及能量代谢的影响。方法取肠上皮细胞株IEC-6分别转染Ad-HSP70腺病毒载体和空腺病毒载体，蛋白质印迹法观察HSP70的表达。取IEC-6细胞分为正常对照组（不作任何处理）、缺氧／复氧组（给予缺氧／复氧处理）、Ad-HSP70转染组（转染Ad-HSP70腺病毒载体后，给予缺氧／复氧处理）。采用噻唑蓝比色法检测细胞内线粒体脱氢酶的活性，高效液相色谱分析法测定细胞能量代谢。结果与转染空腺病毒载体相比，转染Ad-HSP70腺病毒载体可显著增加细胞HSP70的表达。缺氧／复氧组细胞内线粒体脱氢酶的活性明显低于正常对照组（P〈0．01），Ad-HSP70转染组该指标明显高于缺氧／复氧组（P〈0．01）。缺氧／复氧组细胞内腺苷三磷酸含量较正常对照组显著下降，而腺苷二磷酸、腺苷一磷酸含量显著升高；Ad-HSP70转染组细胞能量指标与正常对照组相似（P〉0．05），较缺氧／复氧组有明显改善（P〈0．05或P〈0．01）。缺氧／复氧组细胞能荷为0．615±0．060，明显低于正常对照组（0．748±0．012，P〈0．01）、Ad-HSP70转染组（0．736±0．028，P〈0．01）。结论Ad-HSP70腺病毒载体转染肠上皮细胞可诱导HSP70表达增加，显著提高细胞缺氧／复氧后胞内腺苷三磷酸的含量及细胞能荷，保护线粒体整体功能，提示线粒体是HSP70保护肠上皮细胞缺氧／复氧损伤的主要靶细胞器之一。
Objective To investigate the effect of recombinant adenovirus-mediated heat shock protein 70 （HSP70） on energy metabolism of mitochondria in intestinal epithelial cells （IEC-6） after hypoxia/ reoxygenation injury . Methods IEC-6 cells were transfected with HSP70 recombinant adenovirus vectors （Ad-HSP70） and empty adenovirus vectors. The expression of HSP70 protein was detected by Western blotting. Cultured IEC-6 cells were divided into： control group （without treatment）, hypoxia/reoxygenation group （with challenge of hypoxia/reoxygenation ） and Ad-HSP70 transfection group （with challenge of hypoxia/reoxygenation after Ad-HSP70 transfection）. The activity of mitochondrial dehydrogenase was assessed by MTT method. The contents of cellular ATP, ADP , AMP and energy charge （EC） were determined by high-performance liquid chromatography （HPLC）. Results The expression of HSP70 protein in IEC-6 cells was significantly upregulated after Ad-HSP70 transfection compared with empty adenovirus vector transfection. Compared with that in control group,the activity of mitochondrial dehydrogenase was significantly lowered in IEC-6 cells in hypoxia/ reoxygenation group（ P 〈0.01 ）. The activity of mitochondrial dehydrogenase in Ad-HSP70 transfection group was significantly greater than that in hypoxia/reoxygenation group （ P 〈 0.01 ）. Compared with those in control group,the content of cellular ATP was significantly decreased in hypoxia/reoxygenation group, the contents of cellular ADP and AMP were significantly increased. The above cell energy indices in Ad-HSP70 transfection group was similar to those in control group （ P 〉 0.05 ） , which were ameliorated compared with those in hypoxia/ reoxygenation group （ P 〈0. 05orP 〈0.01 ）. The cellular EC in hypoxia/reoxygenation group （0. 615 ±0. 060） was significantly lower than that in control group （0.748 ±0.012, P 〈0.01） and Ad-HSP70 transfection group （0.736 ±0.028, P 〈0.01）. Conclusion Ad-HSP70 transfectio
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2008年第3期203-206,共4页
Chinese Journal of Burns
关键词
热休克蛋白质类
肠
上皮细胞
能量代谢
线粒体
细胞低氧
Heat shock proteins
Intestines
Epithelial cells
Energy metabolism
Mitochondria
Cell hypoxia
