目的: 优化重组人粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)单纯疱疹病毒注射液(OrienX010)对肿瘤细胞人乳腺癌细胞(michigan cancer foundation-7,MCF-7)的杀伤活性检测方法。方法: 在不同时间加入不同浓度的活性氧(reactive oxygen species,ROS)抑制剂/ROS清除剂,确定对试验影响最小,且能有效去除活性氧的条件。对优化后的方法进行精密度和适用性验证。分别采用优化前、后的方法对同一批号OrienX010样品进行3次肿瘤细胞杀伤活性检测,确定优化前、后方法是否有统计学差异。用不同代次的MCF-7细胞进行试验来确定细胞可使用代次。结果: 在加入CCK-8(Cell Counting Kit-8)试剂前30 min左右,每孔加入50倍稀释的ROS抑制剂/ROS清除剂10 μL为最佳条件。同一试验人员于同1 d 6次测定OrienX010的杀伤活性感染复数(multiplicity of infection,MOI)半数有效量(median effective dose,ED50)的RSD为15.1%,符合可接受标准;2名实验人员于不同日每人重复测定6次OrienX010的MOI ED50的RSD为20.5%,符合可接受标准。对多批次的OrienX010进行肿瘤细胞杀伤活性检测,检测结果均符合质量标准。采用优化前、后的方法对同一批号OrienX010样品进行3次肿瘤细胞杀伤活性检测,t Stat＞t双尾临界值,说明优化前、后2组数据的平均值有显著性差异。MCF-7细胞检测到P 39,检测结果均合格且稳定。结论: 优化后的方法有效消除了ROS对肿瘤细胞杀伤活性检测的影响,该方法具有良好的精密度及适用性,可用于重组人GM-CSF单纯疱疹病毒注射液(OrienX010)的肿瘤细胞杀伤活性检测;优化前、后的方法有统计学差异;MCF-7细胞可使用到P 39。
Objective: To optimize the tumor cell killing activity assay method of recombinant human GM-CSF herpes simplex virus injection (OrienX010) against michigan cancer foundation-7 cells (MCF-7). Methods: Different concentrations of reactive oxygen species (ROS) inhibitor/ROS scavenger were added at different times to determine the condition that had the least influence on the experiment and can effectively remove ROS. The precision and applicability of the optimized method were verified. Tumor cell killing activity of the same batch of OrienX010 samples was tested 3 times by the assay methods before and after optimization, to determine whether there was a statistic difference before and after optimization. Different generations of MCF-7 cell were tested to determine the cell generation that could be used. Results: About 30 min before adding CCK-8 reagent, 10 μL of 50 times diluted ROS inhibitor/ROS scavenger was added to each well as the best condition. The tumor cell killing activity (MOI ED50) of OrienX010 was tested 6 times on the same day by the same analyst, and the results showed that RSD was 15.1%, meeting acceptable standard. On this basis, the same experiment was tested by another analyst on another day, and the results showed that the RSD of 2 analysts was 20.5%, meeting acceptable standard. Multiple batches of OrienX010 were tested and the results met quality standard. Tumor cell killing activity of the same batch number OrienX010 samples was tested three times by the assay methods before and after optimization. t Stat>t two-tailed critical value, indicating significant differences in the mean values of the two groups of data before and after optimization. MCF-7 cell was tested to P 39, and the results were qualified and stable. Conclusion: The optimized method is effective to eliminate the effect of ROS on tumor cell killing activity detection, and the method has good precision and applicability. The optimized method can be used for the tumor cell killing activity detection of recombinant human GM-CSF herpes simplex virus injection (OrienX010). There is statistic difference before and after optimization. MCF-7 cell can be used up to P 39.
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