含有人热休克蛋白基因的重组腺病毒转染乳鼠心肌细胞的实验研究

目的:研究含有人热休克蛋白70(HSP70)基因的重组腺病毒对乳鼠心肌细胞的转染效率以及基因转染后HSP70的表达。方法:体外原代培养乳鼠心肌细胞,以携带增强绿色荧光蛋白(EGFP)基因的腺病毒载体(Ad.EGFP)按不同感染倍数(MOI)体外感染心肌细胞,通过荧光显微镜、流式细胞仪观察所构建的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。结果: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印迹法以及免疫组化染色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。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,并成功地表达HSP70。

The experimental study of transfecting the cultured neonatal rat myocardiocytes with adenovirus vector coding for HSP70 cDNA

Objective: To study the transfection efficiency of the adenovirus vector coding for heat shock protein 70(HSP70) cDNA and the expression of the HSP70 in the cultured neonatal rat myocardiocytes. Methods: The neonatal rat cardiomyocytes were isolated and cultured. Infection ability, efficiency and safety were examined at various titres by fluorescence microscopy and flow cytomtery when cardiomyocytes were infected with Ad.EGFP. The isolated and cultured neonatal rat cardiomyocytes were infected with adenovirus vector coding for HSP70 cDNA in vitro. Protein expression was evaluated by ELISA, Western-blot and immunohistochemical methods. Results: Over 90% of myocardiocytes were infected. The growth of myocardiocytes was not inhibited at MOI 200. ELISA , Western-blot assay and immunohistochemical analysis showed that the transfected myocardiocytes could overexpressed HSP70 in physiological condition. Conclusion: The Ad.HSP70 can infect myocardiocytes safely in vitro with high efficiency and HSP70 increase significantly in the transfected cells.