冷冻复苏对大鼠肝细胞代谢活性的影响

目的研究冷冻复苏对肝细胞代谢活性及细胞色素P450(CYP)mRNA表达调控的的影响,为冷冻复苏肝细胞用于实验研究提供依据。方法采用程序冷冻降温仪冷冻新鲜分离的大鼠肝细胞,1个月后复苏。实时荧光定量PCR法检测细胞CYP1A2,CYP2B1和CYP3A1 mRNA的表达;高压液相色谱-串联质谱法检测细胞内咪达唑仑α-羟基化、双氯芬酸4-羟基化和右美沙芬去甲基化的代谢产物生成量。结果肝细胞冷冻复苏后存活率与新鲜细胞无明显差异,显微镜下可见细胞贴壁并连成片状,细胞核圆而亮。冷冻复苏肝细胞中CYP1A2和CYP2B1 mRNA的诱导表达与新鲜细胞无明显差异,CYP3A1 mRNA无明显表达。与新鲜细胞相比,咪达唑仑α-羟基化代谢产物水平无明显差异;双氯芬酸4-羟基化产物约为1/2;右美沙芬去甲基化产物则增加了1倍。结论冷冻复苏对肝细胞的代谢活性有一定的影响。在药物代谢研究中须考虑冷冻复苏因素对肝细胞代谢活性的影响,以助于对实验结果做出客观和恰当的判断。

Effect of cryopreservation on metabolic activities of hepatocytes

AIM To study the effect of cryopreservation on metabolic activities and cytochrome P450 (CYP) mRNA expression in hepatocytes and provide support for application of cryopreserved hepatocytes in experimental research. METHODS Freshly isolated rat hepatocytes were cryopreserved with rate-controlled freezer, and thawed after 1 month. Real-time quantitative PCR was used to detect expressions of CYP1A2, CYP2B1 and CYP3A1 mRNA, and LC-MS/MS was used to measure contents of metabolites of midazolam-1′-hydrxylation (OH-Mid), diclofenac-4′-hydroxylation (OH-Dic) and dextromethorphan-O-demethylation (Dex) in hepatocytes, respectively. RESULTS There was no significant difference in cell viability between fresh and cryopreserved hepatocytes. The cryopreserved hepatocytes attached and established extensive cell-cell contact, with round and bright nucleus. CYP1A2 and CYP2B1 mRNA expressions induced by β-naphthoflavone and phenobarbital in cryopreserved hepatocytes were similar to that in the fresh primary cells. However, CYP3A1 mRNA expression did not induced by pregnenolone-16α-carbonitrile in cryopreserved hepatocytes. In cryopreserved hepatocytes, the content of OH-Mid was remained as almost the same as the fresh primary hepatocytes, while contents of OH-Dic decreased approximately as a half, and Dex was double as fresh hepatocytes. CONCLUSION Cryopreservation exerts different effects on metabolic activities of hepatocytes. To acquire objective and appropriate results, it is necessary to consider the different influence of cryopreservation on cell metabolic activity in drug metabolic research.