U-2 OS细胞/CCK-8法测定1型单纯疱疹病毒溶瘤活性

目的： 建立U-2 OS细胞/CCK-8比色法，用于测定1型单纯疱疹病毒（herpes simplex virus type 1，HSV-1）溶瘤活性。方法： 选择3株对HSV-1敏感的的细胞株，在细胞培养板上以梯度浓度的HSV-1感染一定时间后，加入CCK-8进行显色，用酶标仪检测后采用四参数曲线法进行量效关系拟合。在选择出量效关系最好的细胞株基础上，对方法的各项实验条件进行优化，并采用活性测定标准品对结果进行校正分析。对建立的方法初步验证准确性和精密度，并用上述方法对3批样品的溶瘤活性进行检测。结果： 优化后的实验采用U-2 OS细胞株，1：4的样品系列稀释比例，5×10⁴ PFU·mL^-1的样品系列稀释初始浓度，每孔2.5×10⁴个细胞的铺板数量，72 h感染时间和4 h CCK-8试剂反应时间等条件进行样品测定，得到的四参数方程拟合的量效关系曲线R²大于0.99，信噪比较高，初步验证结果显示该方法的回收率为113.4%，日间精密度分析的几何变异系数（GCV）为21.8%。3批测试样品的溶瘤活性分别为3.52×10³、3.93×10⁴和1.61×10⁵ U·mL^-1。结论： 建立的U-2 OS细胞/CCK-8法可用于以HSV-1为载体的基因治疗药物溶瘤活性测定。

Objective: To develop an U-2 OS cells/CCK-8 method for oncolytic activity determination of herpes simplex virus 1(HSV-1). Methods: Three candidate cell lines sensitive to HSV-1 were seeded in 96-well cell plates and infected with serial dilutions of HSV-1 for a certain period of time. Then CCK-8 was added and the cell plates were detected on a microplate reader. The four-parameter model was used for dose-effect curve fitting. The cell line showing the best reactivity was selected, and on this basis, the experimental conditions were optimized and the results were calibrated using a reference standard for activity determination. The accuracy and precision of the established method was preliminarily validated. The oncolytic activity of 3 batches of test samples was determinated using the developed method. Results: The optimized method used U-2 OS as test cell line, 5×10⁴ PFU·mL^-1 as initial concentration of test sample and 1:4 as dilution ratio, 2.5×10⁴ cells per well as cell number, 72 h as infection time and 4 h as reaction time of CCK-8. The dose-response curve fitted the four-parameter model with R² being above 0.99 and the signal-to-noise ratio was high. The results showed that the recovery rate was 113.4%, and the inter-day precision was 21.8%. The oncolytic activities of 3 batches of test samples were 3.52×10³ U·mL^-1, 3.93×10⁴ U·mL^-1 and 1.61×10⁵ U·mL^-1, respectively. Conclusion: The developed U-2 OS cell/CCK-8 method can be used to determine the oncolytic activity of HSV-1gene therapy products.

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