阿德福韦在HK-2亚细胞组分中的含量测定及线粒体损伤机制研究

通过建立亚细胞组分药物浓度测定方法,探讨阿德福韦(ADV, adefovir)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 利用免疫磁性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realtimePCR测定线粒体DNA(mtDNA)的相对含量。结果 HK-2细胞线粒体组分中ADV含量为12.5±3.8 ng/mL,显著高于细胞核组分中ADV含量2.3±1.7ng/mL, 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；线粒体动力学蛋白drp1可逆转ADV对HK-2细胞mtDNA的抑制作用。

Determination of Adefovir in HK-2 Subcellular Fragment and Mechanism of Mitochondrial Injury

To investigate the mechanism of adefovir (ADV, adefovir) 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 the mitochondrial fraction of HK-2 cells was 12.5 ± 3.8 ng / mL, which was significantly higher than that of the nucleus in the mitochondrial fraction of mitochondria (P <0.05), and the content of ADV in the mitochondrial fraction of Mdivi-1 was significantly lower The components had no effect (P <0.05). The relative content of mtDNA in ADV was significantly lower than that in blank control group, while Mdivi-1 could reverse this change. Conclusion ADV is mainly distributed in HK-2 cell mitochondria. ADV inhibits mtDNA significantly. The mitochondrial kinetic protein drp1 reverses the inhibitory effect of ADV on mtDNA of HK-2 cells.