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目的探讨龙葵碱对HepG2细胞NAT酶米氏常数Km及最大反应速率Vmax的影响。方法MTT法测定龙葵碱对消化系统SGC-7901人胃癌、HepG2人肝癌、Ls-174人大肠癌3种肿瘤细胞株的细胞毒作用,采用高效液相色谱(HPLC)法,以2-AF为底物,以2-AF的浓度为底物浓度,在以HepG2完整细胞及细胞质内2-AF被NAT酶乙酰化为2-AAF的速度为NAT酶的反应速率,采用双倒数作图法,以底物2-AF浓度的倒数1/S对NAT反应速率的倒数1/V作直线,得出回归方程,计算Km和Vmax。结果MTT法测定表明龙葵碱对HepG2人肝癌细胞比较敏感,酶动力学研究表明,以2-AF为底物,对于HepG2完整细胞,阴性对照组的Km和Vmax分别为(2.37×10-3±8.37×10-5)mmol.L-1、(9.16×10-4±7.54×10-5)nmol.106cells-1,龙葵碱组的Km和Vmax分别为(2.22×10-3±9.05×10-5)mmol.L-1和(5.14×10-4±3.72×10-5)nmol.106cells-1。对于HepG2细胞质,阴性对照组的Km和Vmax分别为(8.95×10-3±2.61×10-4)mmol.L-1、(2.55×10-6±1.92×10-8)μmol.min-1g-1Pro,龙葵碱组的Km和Vmax分别为(9.48×10-3±3.63×10-4)mmol.L-1和(2.43×10-6±1.32×10-8)μmol.min-1g-1Pro,统计学表明对于HepG2完整细胞和细胞质,阴性对照组和龙葵碱组的Km没有差异,而Vmax差异有显著性(完整细胞P<0.01,细胞质P<0.05)。结论龙葵碱是HepG2人肝癌细胞NAT酶2-AF底物的非竞争性抑制剂。
Aim To investigate the effect of solanine on the Km and the maximum reaction rate (Vmax) of Km of NATs in HepG2 cells. Methods MTT assay was used to determine the cytotoxicity of solanine on the three tumor cell lines SGC-7901 human gastric cancer, HepG2 human liver cancer and Ls-174 human colorectal cancer. Using high performance liquid chromatography (HPLC) As the substrate, 2-AF concentration of substrate concentration, in HepG2 intact cells and cytoplasm 2-AF acetylation of NAT enzyme into 2-AAF rate of NAT enzyme reaction rate, using double reciprocal mapping , The reciprocal 1 / V of NAT reaction rate was linearized with the reciprocal 1 / S of substrate 2-AF concentration, the regression equation was obtained and Km and Vmax were calculated. Results MTT assay showed that solanine was more sensitive to HepG2 human hepatoma cells. Kinetic studies showed that the Km and Vmax of HepG2 cells were 2.37 × 10-3 ± 8.37 × 10-5) mmol·L-1, (9.16 × 10-4 ± 7.54 × 10-5) nmol.106cells-1, respectively. The values of Km and Vmax of solanine were (2.22 × 10-3 ± 9.05 × 10-5) mmol.L-1 and (5.14 × 10-4 ± 3.72 × 10-5) nmol.106cells-1. For HepG2 cytoplasm, Km and Vmax of the negative control group were (8.95 × 10-3 ± 2.61 × 10-4) mmol.L-1, (2.55 × 10-6 ± 1.92 × 10-8) μmol · min-1g -1Pro and solanine groups were (9.48 × 10-3 ± 3.63 × 10-4) mmol.L-1 and (2.43 × 10-6 ± 1.32 × 10-8) μmol · min-1g -1Pro. Statistical analysis showed that for HepG2 intact cells and cytoplasm, there was no difference in Km between negative control group and solanine group, but Vmax difference was significant (P <0.01 in intact cells and P <0.05 in cytoplasm). Conclusion Solanine is a noncompetitive inhibitor of NAT enzyme 2-AF substrate in HepG2 human hepatoma cells.