以半乳糖作为生长能源的HepG2细胞线粒体毒性评价模型的建立与应用  

Evaluation model of mitochondrial toxicity in galactose-grown HepG2 cells

作　　者：胡倩倩[1] 谭初兵[2] 张洁[1] 潘晓菲[1] 王士伟[2] 时丽丽[2] 徐为人[2] 

机构地区：[1]天津医科大学基础医学院,天津300070 [2]天津药物研究院新药设计与发现重点实验室,天津300193

出　　处：《中国药理学通报》2013年第11期1617-1621,共5页Chinese Pharmacological Bulletin

基　　金：国家"重大新药创制"科技重大专项(No2012ZX09105102;2011ZX09401-009)

摘　　要：目的针对药源性肝脏线粒体毒性问题,利用人肝癌HepG2细胞分别建立以葡萄糖和半乳糖作为生长能源的线粒体毒性评价模型,并初步应用于核苷类似物药物线粒体毒性的评价。方法采用半乳糖替换葡萄糖作为HepG2细胞生长的能量来源,通过对半乳糖浓度及作用时间的优化,建立线粒体毒性评价模型,并用线粒体抑制剂进行验证,进而评价核苷类似物的线粒体毒性。结果 (1)线粒体抑制剂作用24 h结果显示:葡萄糖模型中,鱼藤酮、寡霉素、CCCP浓度分别在1、10、1μmol·L-1即可见明显的线粒体毒性,而在半乳糖模型中3者均在1 nmol·L-1时已表现出明显的线粒体毒性,抑制率分别为81.8%(P<0.01)、46.4%(P<0.01)、20.0%(P<0.05);相比之下,作用48 h,3者在半乳糖细胞模型中的线粒体功能抑制率比葡萄糖模型中更显著。(2)核苷类似物在葡萄糖模型中未见明显的线粒体毒性。而在半乳糖模型中,药物作用24 h,10μmol·L-1更昔洛韦、喷昔洛韦、阿昔洛韦即表现出明显的线粒体毒性,抑制率分别达7.5%(P<0.05)、6.9%(P<0.05)、8.2%(P<0.05),100μmol·L-1的抑制率更高;药物作用48 h,上述药物的线粒体功能抑制率更高。结论以半乳糖替代葡萄糖作为细胞生长碳源时,可避免细胞Crabtree effect效应,使得线粒体毒性物质作用细胞后线粒体损伤更易显现,尤其对于线粒体低毒的化合物而言,利用半乳糖作为细胞生长碳源使其线粒体毒性更易评价。Aim In allusion to drug-induced liver mitochondrial toxicity problems, the mitochondrial toxicity evaluation model relying on glucose and galaetose through human liver cancer cell HepG2 was built separately, and preliminailily applied to nueleoside analogue drug mitochondrial toxicity assessment. Methods Galaetose was used to replace glucose as the energy source for HepG2 cells. The lactose concentration and effecting time were optimized to establish mitoehondrial toxicity evaluation model, and then the nueleoside analogues of mitoehondrial toxicity were evaluated by mitochoudrial inhibitor validation model. Results （ 1 ） 24 h after mitochondrial inhibitor took effect, rotenone, oligomycin and CCCP showed obvious mitochondrial toxicity at concentrations of 1 , 10, 1 μmol ·L^-1 respectively when glucose as the carbon source, however, obvious mitochondrial toxicity was observed at 1 nmol ·L^-1 when relying on galactose, with 81.8% （P〈0. 01）, 46.4% （P〈0.01）, 20.0% （P〈0. 05） inhibition ratio respectively and became higher if mitochondrial inhibitor effect for 48 h. （2） Nucleoside analogues showed that there was no obvious mitochondrial toxicity in glucose model 24 h and 48 h afler drug effect.While in galactose model, obvious mitochondrial toxicity could be detected by 10 μmol · L^-1 of ganciclovir, pcnciclovir and acyclovir 24 h after drug effect, with 7.5%（P〈0. 05）,6.9%（P〈0.05）,8.2%（P〈0. 05） inhibition ratio respectively, and the ratio of 100 μmol · L-1 was higher, 48 h after drug effect, the tunction inhibition rate of the abore-mentioned drugs was higher. Conclusion When media glucose is replaced with galactose as the carbon source of HepG2 cells, the Crahtree effect could be avoided, and it is easier to present miloehondria damage after toxicity substance effect, and to evaluate the mitochondrial toxicity especially tor mitochondrial low-toxic compound.

关 键 词：线粒体毒性 HEPG2细胞 核苷类似物 半乳糖 线粒 体抑制剂 Crabtree effect ATP 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R342.5[医药卫生—基础医学]