摘要
目的:通过建立亚细胞组分药物浓度测定方法,探讨阿德福韦(adefovir,ADV)对人肾小管上皮细胞株(HK-2)损伤的机制,以及线粒体分裂相关蛋白drp1抑制剂对损伤的保护作用。方法:利用免疫磁选分离法分离亚细胞组分;利用液质联用仪(LC-MS/MS)测定亚细胞组分中的ADV的浓度;利用realtime PCR测定线粒体DNA(mtDNA)的相对含量。结果:ADV处理组HK-2细胞线粒体组分中ADV含量为(9.16±2.78)amol/cell,显著高于细胞核组分中ADV含量(1.68±1.25)amol/cell,经Mdivi-1预处理的线粒体组分中ADV含量显著降低,而细胞核中组分无影响(P<0.05)。ADV干预后mtDNA相对含量显著低于空白对照组,而Mdivi-1可逆转这一变化。结论:ADV主要分布在HK-2细胞线粒体中;ADV对mtDNA明显抑制作用;Mdivi-1可逆转ADV对HK-2细胞mtDNA的抑制作用。
Objective： To investigate the mechanism of adefovir（ADV） damage to human renal tubular epithelial cell line（HK-2） and the protective effect of mitochondrial cleavage-related protein drp1 inhibitor on the damage by establishing the subcellular fraction drug concentration assay. Methods： Subcellular components were isolated by immunomagnetic separation. The concentration of ADV in the subcellular fraction was determined by LC-MS/MS（liquid chromatography-tandem mass spectrometry）. The relative content of mitochondrial DNA（mtDNA） was determined by realtime PCR. Results： The content of ADV in mitochondria of HK-2 cells was（9.16 ±2.78 amol/cell） in ADV-treated group, which was significantly higher than that in the nucleus（1.68±1.25 amol/cell）. The mitochondrial fraction pretreated by Mdivi-1 had a significantly lower level of ADV and no effect on the nuclear fraction（P0.05）. The relative content of mtDNA after ADV intervention was significantly lower than that of the blank control group, while Mdivi-1 could reverse this change. Conclusion： ADV is mainly distributed in HK-2 cell mitochondria. ADV inhibits mtDNA significantly, and the Mdivi-1 can reverse the inhibitory effect of ADV on mtDNA in HK-2 cells.
出处
《药学与临床研究》
2017年第6期481-485,共5页
Pharmaceutical and Clinical Research
基金
南京市医学科技发展资金资助项目(YKK14151)
