乙肝抗病毒药物苦参碱联合拉米夫定对乙肝病毒DNA聚合酶活性的影响

Effect of matrine combined with lamivudine on activity of HBV DNA polymerase

目的:探讨苦参碱联合拉米夫定抗乙肝协同作用的研究及对乙肝病毒DNA聚合酶(HBV DNAP)活性的影响。方法:本文采用MTT法测定苦参碱和拉米夫定对转染人肝癌细胞株(Hep G2)的2.2.15细胞生长抑制能力,利用酶抑制动力学方法,研究了苦参碱和拉米夫定对HBV DNAP的抑制作用、并进一步地分析了苦参碱对HBV DNAP的抑制动力学常数,最后对苦参碱联合拉米夫定对HepG2.2.15细胞生长抑制和HBV DNA合成的抑制是否具有协同效应进行了探讨。结果:苦参碱是一种可逆的竞争型HBV DNAP抑制剂(半抑制浓度IC50为11.24±2.06μmol/L,抑制常数Ki为0.34±0.05μmol/L),圆二色性谱分析表明苦参碱诱导HBV DNAP的构象发生部分改变,α-螺旋含量逐渐增加;且低浓度的拉米夫定与苦参碱联合使用对Hep G2.2.15细胞生长抑制和对HBV DNA合成的抑制都具有协同作用。结论:苦参碱能够通过结合到HBV DNAP的活性中心,并诱导酶的结构变得紧密,而不利于活性中心的形成,最终使HBV DNAP活性降低。而这种诱导酶的活性降低直接影响到乙肝病毒DNA的合成,加之拉米夫定的联合使用,起到了协同作用,增加了抗乙肝病毒的药效。

Objective： To study the synergistic mechanism of matrine combined with lamivudine on hepatitis B virus（ HBV）,and to investigate the effect on DNA polymerase activity of HBV（ HBV DNAP）. Methods： The inhibitory effect of matrine and lamivudine on the growth of HepG2. 2. 15 cells was measured by MTT assay,and the inhibitory effect of matrine and lamivudine on HBV DNAP was detected by an inhibitory kinetic method. The inhibitory kinetic constants of matrine on HBV DNAP was further analyzed,and the synergistic effect of matrine combined with lamivudine on the inhibition of HepG2. 2. 15 cell growth and DNA HBV synthesis was discussed. Results： Matrine had a good inhibitory effect on the growth of HepG2. 2. 15 cells. The half inhibitory concentration（ IC50） was（ 13. 44 ± 2. 11） μmol/L. In addition,matrine was a reversible competitive type HBV DNAP inhibitor. The circular dichroism analysis showed that the content of α-helical gradually increased,and the conformation of HBV DNAP induced by matrine was changed. Low concentration of lamivudine combined with matrine had synergistic effect on the inhibition of HepG2. 2. 15 cell growth and HBV DNA synthesis. Conclusion： The binding of matrine to the active center of HBV DNAP HBV induces the reduction of HBV DNAP activity and closes up the enzyme structure,which is not conducive to the formation of an active center,thus inducing the decrease of DNA synthesis. Matrine combined with lamivudine has a synergistic effect.