PiggyBac介导CYP3A4转基因肝细胞系的长期稳定表达研究及其药物肝毒性评价

发布时间：2018-06-09 11:38

本文选题：药物性肝损伤 + HepG2细胞　；参考：《华南理工大学》2016年硕士论文

【摘要】：在新药研发和临床用药过程中,评估药物性肝损伤是一个重要的命题。原代肝细胞是进行体外肝代谢研究的“金标准”,但因来源稀少、表型不稳定及个体差异显著等原因,其应用受到了很大的限制。来源于肝脏组织的肝癌细胞系则因药物代谢酶表达水平较低而无法准确地预测或评估药物性肝损伤。为了克服这一限制,本研究运用PiggyBac转座子系统构建了稳定高效表达CYP3A4代谢酶的转基因HepG2细胞。通过PiggyBac转座子系统,CYP3A4代谢酶编码基因被成功导入HepG2细胞。结合抗性筛选、流式分选技术、有限稀释法和克隆环技术,本研究获得了20株稳定表达CYP3A4的HepG2单克隆细胞。通过对其中三株单克隆细胞进行mRNA表达、蛋白质表达和代谢活性的鉴定发现,转基因肝细胞系的CYP3A4表达水平和代谢活性相比于野生型HepG2细胞明显提高。选取mRNA相对表达量最高的转基因肝细胞系HepG/PB3A4.10进行持续传代培养,研究其CYP3A4表达的长期稳定性。结果显示,HepG/PB3A4.10持续培养40代后,仍然具有显著高于野生型HepG2细胞的CYP3A4表达水平和代谢活性,且不同代数(P20、P30、P40)转基因肝细胞系间的CYP3A4表达水平和代谢活性并未呈现显著差异。随后,本研究以HepG/PB3A4.10细胞为基础通过MTT法测定两种生物活性化合物(曲格列酮和对乙酰氨基酚)以及两种非生物活性化合物(蔗糖和半乳糖胺)的细胞毒性,发现经过生物活性药物处理后的HepG/PB3A4.10细胞存活率明显减少,显著低于野生型HepG2细胞。此外,与已有的文献报道相比,本研究所构建的转基因肝细胞系对曲格列酮和对乙酰氨基酚的代谢毒性更加敏感。简而言之,本研究利用PiggyBac转座子系统成功快速构建了稳定表达CYP3A4代谢酶的转基因肝细胞系,且所获转基因肝细胞系对CYP3A4介导的药物肝毒性具有高度敏感性。该细胞系为临床前药物研发提供了一个可筛查CYP3A4介导的潜在肝毒性的体外研究模型。与此同时,本研究中采用的PiggyBac转座子方法提示了一种在用于研究药物性肝损伤的体外细胞模型中实现两种或两种以上P450酶长期稳定表达的有效途径。
[Abstract]:Evaluation of drug-induced liver injury is an important proposition in the process of new drug development and clinical use. Primary hepatocytes are the "gold standard" for the study of liver metabolism in vitro, but their application is limited because of their rare sources, unstable phenotypes and significant individual differences. Liver cancer cell lines derived from liver tissues can not accurately predict or evaluate drug-induced liver injury due to the low expression of drug metabolizing enzymes. In order to overcome this limitation, transgenic HepG2 cells expressing CYP3A4 metabolic enzyme stably and efficiently were constructed by PiggyBac transposon system. CYP3A4 metabolic enzyme encoding gene was successfully transfected into HepG2 cells by PiggyBac transposition subsystem. Combined with resistance screening, flow sorting, limited dilution and cloning technique, 20 HepG2 cells stably expressing CYP3A4 were obtained. The expression of CYP3A4 mRNA, protein expression and metabolic activity of three of them were significantly higher than those of wild type HepG2 cells, the expression level and metabolic activity of CYP3A4 in transgenic liver cell lines were significantly higher than those in wild type HepG2 cells. The transgenic liver cell line HepG / PB3A4.10 with the highest relative mRNA expression was selected for continuous passage culture to study the long-term stability of CYP3A4 expression. The results showed that the CYP3A4 expression and metabolic activity of HepG / PB3A4.10 were still significantly higher than those of wild type HepG2 cells after 40 generations of continuous culture, and there was no significant difference in CYP3A4 expression and metabolic activity among different generations of P20P30 P40). Subsequently, the cytotoxicity of two bioactive compounds (trioglitazone and paracetamol) and two non-bioactive compounds (sucrose and galactosamine) were determined by MTT assay based on HepG / PB3A4.10 cells. It was found that the survival rate of HepG / PB3A4.10 cells treated with bioactive drugs was significantly lower than that of wild type HepG2 cells. In addition, the transgenic liver cell lines were more sensitive to the metabolic toxicity of trioglitazone and acetaminophen. In short, a transgenic liver cell line stably expressing CYP3A4 metabolic enzyme was successfully constructed by using the PiggyBac transposition subsystem, and the obtained transgenic liver cell line was highly sensitive to CYP3A4-mediated hepatotoxicity. The cell line provides an in vitro model for screening CYP3A4 mediated potential hepatotoxicity for preclinical drug development. At the same time, the PiggyBac transposon method used in this study suggests an effective way to achieve the long-term stable expression of two or more P450 enzymes in a drug-induced liver injury model in vitro.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575

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