人源化肝细胞共培养系统为基础的代谢活化系统研究

Metabolic activation system based on Co-culture system of human hepatocytes

目的从不同组合的人源化肝细胞系共培养模型中构建并筛选优势共培养体系,为人源化代谢活化系统在遗传毒性和致突变性中的应用提供理论基础。方法以HepG2/C3A人源肝癌细胞作为代谢活化系统的支持细胞,应用穿孔法对HepG2/C3A、LX-2肝星形细胞和Caco-2人源肠腺癌细胞三细胞进行不同组合的共培养模型的构建和筛选,CCK-8法测定模型中每个细胞的4 d生长曲线以确定能稳定生长的共培养模型,RT-qPCR测定在不同类型培养模型中HepG2/C3A细胞CYP3A4、CYP2E1、CYP1A2、CYP2B6、CYP2C9、GSTA1/2和UGT1A1基因的表达情况。结果由HepG2/C3A-Caco2-LX2细胞构建的三细胞共培养(COL)模型中各细胞生长状态稳定。RT-qPCR结果显示在不同的共培养模型中,肝细胞的Ⅰ相及Ⅱ相代谢酶基因的表达差异较大,COL模型中HepG2/C3A细胞CYP1A2、CYP2C9、CYP3A4、UGT1A1和GSTA1/2基因表达水平分别是单培养组的3.41、4.20、2.88、4.75和5.11倍,差异有统计学意义(P<0.05)。双细胞共培养模型中HepG2/C3A-Caco2(CO-C)和HepG2/C3A-LX2(CL)的肝细胞仅CYP2E1或GSTA1/2相对单培养组表达略有升高,差异有统计学意义(P<0.05),CYP3A4、CYP1A2、CYP2B6和UGT1A1基因的表达水平均有所降低,差异有统计学意义(P<0.05)。结论不同细胞共培养模型对肝细胞的Ⅰ相及Ⅱ相代谢酶基因的表达产生较大影响,相比本研究中的双细胞共培养体系,HepG2/C3A-Caco2-LX2三细胞共培养体系可更稳定的生长,且主要的Ⅰ相及Ⅱ相代谢酶基因表达水平明显升高,有望作为代谢活化系统的优势共培养体系。

Objective The dominant co-culture system was constructed and screened from the co-culture models of different combinations of humanized hepatocytes, which provided a theoretical basis for the application of humanized metabolic activation system in genotoxicity and mutagenicity. Methods HepG2/C3 A human liver cancer cells were used as the supporting cells of the metabolic activation system. HepG2/C3 A, LX-2 hepatic astrocytic and Caco-2 human intestinal adenocarcinoma cells were co-cultured in different combinations using the perforation method. CCK-8 method was used to determine the 4-day growth curve of each cell in the model to determine the expression of the co-culture model that could grow stably. Expression of CYP3 A4, CYP2 E1, CYP1 A2, CYP2 B6, CYP2 C9, GSTA1/2 and UGT1 A1 genes in HepG2/C3 A cells in different types of culture models were determined by RT-qPCR. Result The growth states of each cell were stable in a three-cell co-culture(COL) model constructed from HepG2/C3 A-Caco2-LX2. The result of RT-qPCR showed that the expressions of the genes of phase-Ⅰ and phase-Ⅱ metabolic enzyme in hepatocytes were significantly different in different co-culture models(the expression levels of CYP1 A2、CYP2 C9、CYP3 A4、UGT1 A1 and GSTA1/2 in HepG2/C3 A cells in COL model were 3.41, 4.20, 2.88, 4.75, 5.11 times of the monoculture group, respectively, with statistical significance(P<0.05). Hepatocytes of HepG2/C3 A-Caco2 and HepG2/C3 A-LX2 in a two-cell co-culture model showed only slightly higher expression of CYP2 E1 or GSTA1/2 than in a single culture group,with statistical significance(P<0.05). The expression levels of CYP3 A4、CYP1 A2、CYP2 B6 and UGT1 A1 were all decreased with statistical significance(P<0.05). Conclusion The expressions of phase-Ⅰ and phase-Ⅱ metabolizing enzyme genes in hepatocytes were influenced by different co-culture models. Compared with the two-cell co-culture system in this study, HepG2/C3 A-Caco2-LX2 three-cell co-culture system can be more stable growth, and the main i-phase and ii-phase metabolic enzyme gene expression levels are significantly increased, which is expected to be the dominant co-culture system of metabolic activation system.