体外遗传毒性评价试验的优化

发布时间：2018-09-11 08:18

【摘要】：遗传毒性试验可以考察受试物是否具有潜在致癌性或者致突变性，是药物毒性评价和环境致癌物研究的重要组成部分。建立一个具有良好特异性和敏感性并且操作简便的体外试验体系，可以在药物早期研发过程中提供重要的参考依据，降低研发风险。此外，在体外实验体系中采用合理的代谢活化条件，可以更加科学地评价化合物的遗传毒性。我们应用人淋巴瘤母细胞（TK6）建立了优化的遗传毒性组合试验体系。在同一处理条件下的不同时间点对受试物分别进行彗星试验，体外微核试验和TK基因突变试验。该体系的优点为TK6细胞为人源细胞，与常用的小鼠细胞相比，，p53基因表达正常；此外，体系具有良好特异性与敏感性，操作简便；该检测方法覆盖了DNA损伤、染色体异常和基因突变三个遗传终点。在本研究中我们运用该试验体系考察了中药单体——汉防己甲素和雷公藤甲素的遗传毒性。彗星试验结果显示汉防己甲素可能导致DNA的断裂，体外微核试验表明雷公藤甲素可能导致染色体损伤，提示两者都具有潜在的遗传毒性。在致癌物代谢活化中，细胞色素P450酶CYP1A1和CYP2E1是常见的两种代谢酶。本研究应用可稳定表达CYP1A1的AHH-1细胞株和同时稳定表达CYP1A1和CYP2E1的h2E1V2细胞株，建立了体外TK基因突变实验体系，考察了环境致癌物丙烯酰胺通过CYP1A1和CYP2E1代谢后的遗传毒性。试验结果表明，丙烯酰胺对h2E1V2细胞具有致突变作用，表明CYP2E1在其代谢活化中发挥重要作用。综上所述，本研究建立了两个优化哺乳动物细胞的遗传毒性试验组合体系，具有良好特异性、敏感性，并且操作简便。对两种中药单体受试物和一种环境致癌物进行了毒性评价和初步机制探讨，验证了实验体系并得到了毒性评价结果。
[Abstract]:Genotoxicity test is an important part of drug toxicity evaluation and environmental carcinogen research, which can be used to investigate the potential carcinogenicity or mutagenicity of the tested material. The establishment of an in vitro test system with good specificity and sensitivity and simple operation can provide an important reference for early drug development and reduce R & D risk. In addition, the genetic toxicity of the compounds can be evaluated more scientifically by using reasonable metabolic activation conditions in vitro. We have established an optimized genotoxicity combination test system using human lymphoma mother cells (TK6). Comet assay in vitro micronucleus test and TK gene mutation test were carried out at different time points under the same treatment conditions. The advantages of this system are that TK6 cells are derived from human cells, and the expression of p53 gene is normal compared with common mouse cells. In addition, the system has good specificity and sensitivity, and is easy to operate. The detection method covers DNA damage. Chromosomal abnormalities and gene mutations are three genetic endpoints. The genotoxicity of Tetrandrine and Tripterygium wilfordii were investigated by using this experimental system. Comet assay showed that Tetrandrine might lead to DNA breakage. Micronucleus test in vitro indicated that triptolide might cause chromosome damage, suggesting that both of them have potential genotoxicity. Cytochrome P450 enzyme CYP1A1 and CYP2E1 are two common metabolic enzymes in the metabolic activation of carcinogens. In this study, AHH-1 cell lines expressing CYP1A1 stably and h2E1V2 cells expressing CYP1A1 and CYP2E1 stably at the same time were used to establish TK gene mutation assay system in vitro. The genotoxicity of environmental carcinogen acrylamide via CYP1A1 and CYP2E1 metabolism was investigated. The results showed that acrylamide had mutagenicity on h2E1V2 cells, suggesting that CYP2E1 played an important role in the activation of CYP2E1 metabolism. In conclusion, two optimal genetic toxicity test systems for mammalian cells were established, which were specific, sensitive and easy to operate. The toxicity evaluation and preliminary mechanism of two Chinese medicine monomers and one environmental carcinogen were carried out. The experimental system was verified and the results of toxicity evaluation were obtained.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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