基于实时无标记细胞分析技术时间维度特征的体外抗肿瘤活性评价新策略

A new strategy for evaluating antitumor activity in vitro with time-dimensional characteristics of RTCA technology

目的基于实时无标记细胞分析(real time cellular analysis,RTCA)技术,以黄芩苷、黄芪甲苷、橙皮苷和顺铂为例进行抗A549、H1299肺腺癌活性分析,建立一种反映时间维度变化特征的EC_(50)评价新策略。方法应用RTCA Software Pro数据分析及GraphPad Prism、Origin Pro做图,分别采用终点法和时间维度表征黄芩苷、黄芪甲苷、橙皮苷、顺铂受试液/药的体外抗A549、H1299肺腺癌活性。结果①受试液/药对A549及H1299细胞在24 h、48 h终点法中的EC_(50)值有的存在较大差异。②采用四参数方程拟合曲线的相关系数>0.9,EC_(50)动态变化相对稳定期(相邻4点EC_(50)线性拟合|斜率|≤1)计算此时间维度内的EC_(50)值。黄芩苷、黄芪甲苷、橙皮苷和顺铂对A549细胞的EC_(50)分别为52.97±1.75μmol·L^(-1)(16~48 h)、62.88±2.91μmol·L^(-1)(32.25~48 h)、78.84±0.33μmol·L^(-1)(21.5~29.75 h)、13.57±1.54μmol·L^(-1)(27.5~48 h);黄芩苷、黄芪甲苷、橙皮苷和顺铂对H1299细胞的EC_(50)分别为43.71±1.26μmol·L^(-1)(19.5~48 h)、47.23±1.19μmol·L^(-1)(14~48 h)、39.45±0.24μmol·L^(-1)(12.75~46.25 h)、25.97±4.76μmol·L^(-1)(10.25~48 h)。显示受试液/药抗肿瘤起效的时间窗口期不同。结论基于RTCA技术,选用拟合度好、稳定并带有时间维度特征的EC_(50)数据用于抗肿瘤活性评价将更加精确和合理。此外,这种区分开不同有效时间的抗肿瘤药物方法可为临床联合用药的给药时机提供参考,本思路也将为其他体外相关研究提供借鉴。

Aim To analyze the anti-A549 and H1299 lung adenocarcinoma activities via using examples of baicalin,astragaloside,hesperidin and cisplatin based on real time cellular analysis(RTCA)technology,and to build a new strategy for EC_(50)evaluation reflecting the time-dimensional characteristic.Methods Using RTCA Software Pro for data analysis and GraphPad Prism and Origin Pro plotting,the in vitro anti-A549 and H1299 lung adenocarcinoma activities of baicalin,astragaloside,hesperidin,and cisplatin were characterized using the endpoint method and time dimension,respectively.Results①There were significant differences in EC_(50)values of A549 and H1299 cells at 24 h and 48 h endpoint methods.②The correlation coefficient of the curve fitted with the four-parameter equation was>0.9,and the dynamic change of EC_(50)remained relatively stable(the linear fitting of EC_(50)at adjacent 4 points|slope|≤1)used to calculate the EC_(50)value within this time dimension.The EC_(50)of baicalin,astragaloside,hesperidin and cisplatin on A549 cells was 52.97±1.75μmol·L^(-1)(16~48 h),62.88±2.91μmol·L^(-1)(32.25~48 h),78.84±0.33μmol·L^(-1)(21.5~29.75 h),13.57±1.54μmol·L^(-1)(27.5~48 h),respectively;the EC_(50)of baicalin,astragaloside,hesperidin and cisplatin on H1299 cells was 43.71±1.26μmol·L^(-1)(19.5~48 h),47.23±1.19μmol·L^(-1)(14~48 h),39.45±0.24μmol·L^(-1)(12.75~46.25 h),25.97±4.76μmol·L^(-1)(10.25~48 h),respectively.The results showed that the time window for the anti-tumor effect of the test solution/drug was different.Conclusions Based on RTCA technology,it is more accurate and reasonable to select EC_(50)data that exhibit better fitting,stable changes,and time-dimensional characteristics for the evaluation of anti-tumor activity.In addition,this method of distinguishing different effective time of antitumor drugs can provide a reference for the timing of clinical combination drugs,and this approach will also provide a reference for further related studies.