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目的 :探讨不同温度对重组腺病毒Ad_HGF保存时间的影响。方法 :以含 10 %甘油的Hanks液保存Ad_HGF(携带人肝细胞生长因子基因 )、Ad_GFP(携带绿色荧光蛋白基因 ) ,用快速细胞病变 (CPE)法、ELISA方法、流式细胞仪分别检测在不同贮存温度 (- 6 0℃ ,- 2 0℃ ,4℃ ,2 5℃和 37℃ )、不同保存时间的重组腺病毒对其感染滴度、目的基因表达和转染细胞效率的影响。结果 :- 6 0℃保存最长时间 4 4个月 ,其转染效率、HGF蛋白表达、病毒滴度未见明显下降 ;- 2 0℃保存 12个月各指标未见明显变化 ;4℃冰箱保存 2个月 ,其各项指标已有所下降 ,但不是很明显 ,而保存 7个月时 ,其转染效率、HGF蛋白表达量及病毒滴度均明显下降 ;2 5℃和 37℃保存 ,除病毒滴度外 ,其余指标呈逐日下降趋势。结论 :该病毒保存于 - 2 0℃以下性能是稳定的 ,可以满足临床贮存和应用条件。
Objective: To investigate the effect of different temperature on the preservation of recombinant adenovirus Ad_HGF. Methods: Ad_HGF (carrying human hepatocyte growth factor gene) and Ad_GFP (carrying green fluorescent protein gene) were stored in Hanks’ solution containing 10% glycerol and detected by using rapid cytopathic effect (CPE), ELISA and flow cytometry The effects of recombinant adenoviruses at different storage temperatures (-60 ℃, -20 ℃, 4 ℃, 25 ℃ and 37 ℃) on the titers of infection, gene expression and cell efficiency were analyzed. Results: After the longest time of 4 months was stored at 60 ℃, the transfection efficiency, HGF protein expression and virus titer did not decrease significantly. No obvious changes were observed after 12 months of storage at 20 ℃. After 2 months of storage, its indexes have decreased, but it is not obvious. However, the transfection efficiency, HGF protein expression and virus titer dropped significantly after storage for 7 months; and were preserved at 25 ℃ and 37 ℃ In addition to the virus titer, the remaining indicators showed a downward trend day by day. Conclusion: The performance of the virus stored at -20 ℃ is stable, and can meet the clinical storage and application conditions.