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目的建立铜绿假单胞菌注射液(Pseudomonas aeruginosa,PA-MSHA)体外抗肿瘤生物活性检测方法。方法将系列稀释的PA-MSHA在体外直接作用于人肝癌细胞BEL-7402,光学显微镜下观察细胞的生长状态;MTT比色法检测其对细胞增殖水平的影响;对10批PA-MSHA成品分别检测3次,以半数抑制浓度(IC50)为指标,计算批内和批间变异系数(CV),验证方法的精密性。结果随着PA-MSHA稀释度的降低,光镜下贴壁的BEL-7402细胞数量逐渐减少,细胞皱缩并破碎;MTT比色结果显示,随着PA-MSHA稀释度的降低,孔内颜色逐渐变浅,BEL-7402细胞增殖抑制率逐渐升高,呈一定的剂量-效应关系;10批PA-MSHA成品3次检测的IC50值范围为(3.584～7.778)×108个/ml,批内变异系数为4.58%～24.17%,批间变异系数15.08%～16.95%,总变异系数为15.81%,均符合细胞实验变异系数应小于30%的标准;PA-MSHA成品经该方法检测后,得到的IC50值在(3.109～7.383)×108个/ml范围内时,表明成品的生物活性合格。结论建立了PA-MSHA体外抗肿瘤生物活性检测方法,该方法稳定性好、精密性高,而且具有操作简便、节约成本和时间等优点,可用于对PA-MSHA以及其他类似细菌类生物制品的体外生物活性研究。
Objective To establish an in vitro antitumor bioassay for detecting Pseudomonas aeruginosa (PA-MSHA). Methods A series of diluted PA-MSHA was used to directly affect the growth of human hepatoma BEL-7402 in vitro. The growth of cells was observed under a light microscope. The effect of PA-MSHA on the proliferation of the cells was evaluated by MTT colorimetric assay. After three times of detection, the intra-and inter-assay coefficient of variation (CV) was calculated using half-maximal inhibitory concentration (IC50) as an indicator to verify the accuracy of the method. Results As the dilution of PA-MSHA decreased, the number of BEL-7402 cells adhered to each other gradually decreased and the cells collapsed and broken. The colorimetric results of MTT showed that with the decrease of PA-MSHA dilution, The proliferation inhibition rates of BEL-7402 cells gradually increased with a dose-effect relationship. The IC50 values of three batches of 10 batches of PA-MSHA products ranged from (3.584 to 7.778) × 108 cells / ml within the batch The coefficient of variation was 4.58% -24.17%, the coefficient of variation between batches was 15.08% -16.95%, and the total coefficient of variation was 15.81%, which all met the criterion that the coefficient of variation of cell experiment should be less than 30%. After PA-MSHA finished product was tested by this method, The IC50 values ranged from (3.109 ~ 7.383) × 108 / ml, indicating that the biological activity of the finished product was qualified. Conclusion The method for detecting the antitumor biological activity of PA-MSHA in vitro is established. The method has the advantages of good stability and high precision, and has the advantages of simple operation, cost saving and time saving, and can be used for detecting PA-MSHA and other similar bacterial biological products In vitro biological activity.