| 摘要: |
| 目的:研究热休克蛋白70（HSP70）基因转染对体外培养的乳鼠心肌细胞缺氧/复氧损伤的保护作用。方法:体外原代培养乳鼠心肌细胞,以携带增强型绿色荧光蛋白（EGFP）基因的腺病毒载体（Ad.EGFP）按不同感染倍数（MOI）感染体外心肌细胞,通过荧光显微镜、流式细胞仪观察所构建腺病毒的感染力和安全性；应用含有人HSP70基因的重组腺病毒（Ad.HSP70）进行体外感染,采用ELISA法、Western印迹法及免疫组化染色法检测HSP70基因在心肌细胞中的表达情况。利用体外心肌细胞缺氧/复氧损伤模型,研究HSP70基因转染对心肌细胞的保护作用。结果:原代培养获得高纯度的乳鼠心肌细胞,随着MOI值升高,Ad.EGFP的感染效率和基因表达增强,在MOI值为50时,Ad.EGFP感染心肌细胞的效率高于90%,在MOI值为200时,未见心肌细胞的生长明显受抑；ELISA法、Western印迹法证实转染的心肌细胞在正常生理状态下,可高水平表达HSP70。免疫组化染色法结果显示,表达的HSP70主要分布于心肌细胞的胞质和胞核中；利用体外的缺氧/复氧损伤模拟在体的缺血/再灌注损伤,结果显示Ad.HSP70感染组的细胞活力、MTT代谢率、细胞凋亡率、心肌酶谱的变化等指标均优于对照组（P<0.05）。结论:重组腺病毒Ad.HSP70能够在体外高效、安全地感染心肌细胞,并成功表达HSP70；HSP70高表达对体外培养的乳鼠心肌细胞缺氧/复氧损伤具有显著的保护作用。 |
| 关键词: 热休克蛋白70 基因转染 心肌细胞 腺病毒载体 缺氧/复氧损伤 |
| DOI：10.3724/SP.J.1008.2009.01344 |
| 投稿时间：2009-07-19修订日期：2009-09-20 |
| 基金项目:南通大学创新基金. |
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| Protective effect of adenovirus-mediated heat shock protein 70 gene transfection against hypoxia/reoxygenation injury of cultured neonatal rat myocardiocytes |
| YU Wei1,YAN Yu2,ZHANG Yu-dong2,NI Qing1* |
| (1.Department of General Surgery,The First People’s Hospital of Yangzhou,Yangzhou 225200,China;2.Department of Cardiothoracic Surgery,Affiliated Hospital of Nantong University,Nantong 226001) |
| Abstract: |
| Objective:To study the protective effect of adenovirus-mediated heat shock protein 70 (HSP70) gene transfection against hypoxia/reoxygenation injury of cultured neonatal rat myocardiocytes.Methods: The neonatal rat cardiomyocytes were isolated,cultured,and transfected with Ad.EGFP.The transfection efficiency and safety were examined at various titrations by fluorescence microscope and flow cytometer.The cultured cardiomyocytes were then infected with adenovirus vector harboring HSP70 cDNA.Protein expression of HSP70 was evaluated by ELISA,Western blotting analysis and immunohistochemical methods.The cytoprotective effect of adenovirus-mediated HSP70 gene transfection was studied using in vitro myocardiocyte hypoxia/reoxygenation model.Results: High purity neonatal rat cardiomyocytes were obtained by primary culture.The infection efficiency and the EGFP expression increased with the increase of MOI.Over 90% of myocardiocytes were infected at MOI 50.The growth of myocardiocytes was not inhibited at MOI 200.ELISA and Western blotting analysis showed that the transfected myocardiocytes overexpressed HSP70 under normal physiological condition.Immunohistochemical analysis showed prominent overexpression of HSP70 in the cytoplasm and nuclei in HSP70 transfection group.Results of hypoxia/reoxygenation injury model showed that the cell vitality,MTT metabolism,cell apoptosis rate,and changes of myocardial enzyme spectrum were significantly improved in the gene transfection group compared with the control group (P<0.05).Conclusion: Recombinant Ad.HSP70 can efficiently and safely infect myocardiocytes in vitro,leading to expression of HSP70 protein.Overexpression of HSP70 induced by adenovirus-mediated transfection has prominent protective effect against hypoxia/reoxygentaion injury of neonatal rat myocardiocytes. |
| Key words: heat shock protein 70 gene transfection myocardiocyte adenovirus vector hypoxia/reoxygenation injury |
