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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。
Objective: To study the protective effect of heat shock protein 70 (HSP70) gene transfection on hypoxia / reoxygenation injury of cultured neonatal rat cardiomyocytes. Methods: Primary cultured neonatal rat cardiomyocytes were infected with adenovirus vector (Ad.EGFP) carrying enhanced green fluorescent protein (EGFP) gene at different multiplicity of infection (MOI). Cardiomyocytes were harvested by fluorescence microscopy, flow cytometry The infectivity and safety of constructed adenovirus were observed. In vitro infection was induced by recombinant adenovirus containing human HSP70 gene (Ad.HSP70). The expression of HSP70 gene was detected by ELISA, Western blotting and immunohistochemistry in cardiomyocytes In the expression of the situation. In vitro cardiomyocytes hypoxia / reoxygenation injury model study HSP70 gene transfection on cardiomyocyte protective effect. RESULTS: Primary cultured neonatal rat cardiomyocytes were obtained. The infection efficiency and gene expression of Ad.EGFP were enhanced with the increase of MOI. At the MOI value of 50, the efficiency of Ad.EGFP infection of cardiomyocytes was higher than 90 %, At the MOI value of 200, no significant inhibition of myocardial cell growth; ELISA, Western blot confirmed that transfected cardiomyocytes in normal physiological state, the high level of expression of HSP70. The results of immunohistochemistry showed that the expression of HSP70 mainly distributed in the cytoplasm and nucleus of cardiomyocytes. In vitro hypoxia / reoxygenation injury mimics ischemia / reperfusion injury in vivo, and the results showed that Ad.HSP70 infection The indexes of cell viability, MTT metabolic rate, apoptosis rate and myocardial enzymogram were all better than the control group (P <0.05). CONCLUSION: The recombinant adenovirus Ad.HSP70 can efficiently and safely infect cardiomyocytes in vitro and successfully express HSP70. High expression of HSP70 has a significant protective effect on hypoxia / reoxygenation injury in cultured neonatal rat cardiomyocytes.