微量过氧乙酸溶液细胞毒性研究

STUDY ON CYTOTOXICITY OF MICRODOSIS PERACETIC ACID IN VITRO

目的评价微量过氧乙酸的细胞毒性,为过氧乙酸灭菌时残余量限值的制定提供实验依据。方法采用体外细胞培养技术,观察体积分数(V/V)为1×10-6、2×10-6、3×10-6、4×10-6、5×10-6和10×10-6的过氧乙酸对第3代L929小鼠成纤维细胞生长的影响,将不含过氧乙酸的培养基作为对照组。分别于培养后2、4和7d经MTT比色法测定各组吸光度(A)值,绘制细胞生长曲线,计算各组细胞相对增殖率,对不同浓度过氧乙酸进行细胞毒性评价。结果培养后2、4和7d,1×10-6组与对照组细胞形态及生长状况相似,其余各组细胞生长较差。MTT法检测显示,各时间点对照组A值最大,除1×10-6组与对照组比较差异无统计学意义(P>0.05)外,其余各浓度组与对照组比较差异均有统计学意义(P<0.05)。相关分析显示,各时间点随着过氧乙酸浓度增加A值逐渐下降,细胞增殖与过氧乙酸浓度成负相关;培养后2、4和7d,相关系数分别为—1.000、—1.000、—0.964(P<0.001),相关程度随时间延长逐渐减小。1×10-6组与对照组生长曲线近似,具有明显的指数增长期,其余组生长曲线较平缓。1×10-6组过氧乙酸的细胞毒性分级为1级,2×10-6组为2级,3×10-6组为3级,4×10-6组为3～4级,5×10-6、10×10-6组均为4级。结论 1×10-6过氧乙酸无明显细胞毒性,浓度超过1×10-6的过氧乙酸具有不同程度细胞毒性。

Objective To evaluate the cytotoxicity of microdosis peracetic acid （PAA） so as to provide the evidence for making residual limit of PAA sterilization. Methods Mouse fibroblasts （L929 cell line） cultured in vitro were observed to evaluate the influence of microdosis PAA including 1 × 10-6 , 2 × 10-6 , 3 × 10-6 , 4 × 10-6 , 5 × 10-6 , and 10 × 10-6 （V/V）. The proliferation of cells was determined by MTT assay at 2, 4, and 7 days of culture. The growth curve and the relative growth rate （RGR） were obtained. The cytotoxicity of PAA at different concentrations was evaluated according to RGR. Results At 2, 4, and 7 days after culture, fibroblasts of 1 × 10-6 group grew with normal morphology analogous to control group, while the cell growth of other groups were poor. With the increase of PAA concentration, the absorbance （A） values decreased, which suggested that there was a significant negative correlation between cell proliferation and PAA concentration. And the correlation coefficient was — 1.000 at 2 and 4 days, — 0.964 at 7 days. There was no significant difference in A value between 1 × 10-6 group and the control group （P 0.05）, while there were significant differences in A value between the control group and other concentration groups （P 0.05）. The growth curve of 1 × 10-6 group was similar to that of the control group, both had obvious phase of exponential growth. The growth curves of other groups had no obvious phase of exponential growth. The cytotoxicity of 1 × 10-6 group was classified as level 1, 2 × 10-6 group as level 2, 3 × 10-6 group as level 3, 4 × 10-6 group as level 3-4, 5 × 10-6 group and 10 × 10-6 group as level 4. Conclusion PAA of 1 × 10-6 had no obvious cytotoxicity. The residual limit of PAA less than 1 × 10-6 was recommended.