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该文采用MTT法测定丹参素对转染人肝癌细胞株(Hep G2)的2.2.15细胞活性的抑制作用,通过间接荧光标记法测定细胞内活性氧水平的变化;采用ELISA法测定细胞上清乙肝表面抗原(HBs Ag)和E抗原(HBe Ag)水平,采用荧光定量PCR法检测HBV DNA水平;利用酶抑制动力学方法,研究了丹参素对HBV RT的抑制作用;最后利用圆二色谱法监测丹参素对HBV逆转录酶二级结构的影响。结果显示丹参素能够对Hep G2.2.15细胞的生长起到良好的抑制作用,半抑制浓度IC50为(15.35±2.43)μmol·L-1;丹参素能显著抑制HBs Ag和HBe Ag表达,同时对HBV DNA复制具有抑制作用;此外,丹参素是一种有效的HBV逆转录酶抑制剂[半抑制浓度IC50为(21.32±2.43)μmol·L-1],荧光标记法检测,细胞内活性氧水平随着丹参素呈浓度依赖性升高;圆二色谱分析表明丹参素诱导HBV逆转录酶的构象发生部分改变,α-螺旋含量逐渐增加。结果表明丹参素能够通过结合HBV逆转录酶,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逆转录酶活性降低。而这种诱导酶的活性降低直接影响到乙肝病毒DNA的复制,加之抗原水平的降低,增加了丹参素抗乙肝病毒的药效。
In this paper, MTT assay was used to determine the inhibitory effect of Danshensu on the activity of 2.2.15 cells transfected with human hepatoma cell line (Hep G2). The changes of intracellular reactive oxygen species (ROS) levels were measured by indirect fluorescent labeling. HBsAg and HBeAg were detected by real-time quantitative PCR. HBV DNA levels were detected by real-time quantitative PCR. The inhibitory effect of Danshensu on HBV RT was studied by enzyme inhibition kinetics. Finally, Effect of Danshensu on Secondary Structure of HBV Reverse Transcriptase. The results showed that Danshensu could effectively inhibit the growth of Hep G2.2.15 cells with the IC50 of (15.35 ± 2.43) μmol·L-1; Danshensu could significantly inhibit the expression of HBsAg and HBeAg, In addition, Danshensu is a potent inhibitor of HBV reverse transcriptase (IC50 of half-inhibitory concentration was (21.32 ± 2.43) μmol·L-1]. Fluorescence labeling assay showed that intracellular reactive oxygen species Danshensu increased in a concentration-dependent manner. Circular dichroism analysis showed that the conformation of HBV reverse transcriptase was partially changed by danshensu, and the content of α-helix increased gradually. The results show that Danshensu can bind HBV reverse transcriptase, inducing enzyme structure becomes tight, but not conducive to the formation of the active center, and ultimately make HBV reverse transcriptase activity decreased. And this activity of induced enzymes directly affect the replication of hepatitis B virus DNA, combined with the reduction of antigen levels, increased the efficacy of Danshensu anti-hepatitis B virus.