血液灌流器体外细胞毒性和遗传毒性试验方法研究

Cytotoxicity and Genotoxicity Test Method of Hemoperfusion

目的:研究一种适合于评价血液灌流器的细胞毒性和遗传毒性试验的方法。方法:选择含血清培养基作为浸提介质,采用灌注的方式分别在37℃振荡浸提24h和48h制备浸提液,然后用新鲜含血清培养基进行稀释,得到100%(未稀释)、50%、25%和12.5%浓度的浸提液,采用MTT法对其进行细胞毒性试验,分别采用体外哺乳动物细胞染色体畸变试验和体外哺乳动物细胞基因突变试验检测样品浸提液的遗传毒性。结果:细胞毒性试验:(1)采用灌注方式制备浸提液可行;(2)浸提原液(100%浓度)因其营养成分被吸附,会造成细胞毒性假阳性结果;(3)浓度为25%和12.5%的浸提液因其稀释倍数过大,浸提液中的毒性成分被稀释,会出现细胞毒性假阴性结果;(4)浓度为50%的浸提液适合细胞毒性评价,其既补充了营养成分,又不至于过度稀释,并且此浓度下的生理盐水对照的细胞存活率>80%,阳性对照的细胞存活率<30%,符合试验体系的要求。遗传毒性试验:(1)体外哺乳动物细胞基因突变试验中各个梯度相对存活率在60%~120%;(2)体外哺乳动物细胞染色体畸变试验中各个梯度染色体畸变率与阴性对照组相比在统计学上均无显著差异性。结论:该试验方法可用于灌流器的细胞毒性试验和遗传毒性试验。

Objective:To study a suitable method for evaluating cytotoxicity and genotoxicity of hemoperfusion.Methods:In this paper,serum containing medium was selected as the extraction medium,and the perfusion method was adopted to prepare the extract by shaking culture at 37˚C for 24h and 48h,respectively.Then,the extract was diluted with fresh serum containing medium,and the concentration of 100%(undiluted),50%,25%and 12.5%of the extract was obtained.The cytotoxicity test was conducted by MTT method.The genotoxicity of the sample extract was detected by In vitro mammalian cell gene mutation test using mouse lymphoma cells and In vitro mammalian cell chromosome aberration test.Results:Cytotoxicity:①It is feasible to prepare the extract by perfusion;②The extract solution(100%concentration)will cause false positive results of cytotoxicity because its nutrients are adsorbed;③Due to excessive dilution times of extracts with 25%and 12.5%concentration,toxic components in the extracts will be diluted,resulting in false negative results of cytotoxicity;④The concentration of 50%extract is suitable for cytotoxicity evaluation,and it supplements nutrients without excessive dilution.Moreover,the cell survival rate of normal saline control at this concentration is greater than 80%,and that of positive control is less than 30%,which meets the requirements of the test system.Genotoxicity:①The relative survival rates of each group in the gene mutation test of mammalian cells in vitro ranged from 60%to 120%;②In the chromosomal aberration test of mammalian cells in vitro,there was no significant difference in the chromosomal aberration rate of each group compared with the negative control group.Conclusion:This method can be used to test the cytotoxicity and genotoxicity of hemoperfusion.