丹参素靶向乙肝病毒逆转录酶抗乙肝及抗原表达的影响机制的研究.docVIP

丹参素靶向乙肝病毒逆转录酶抗乙肝及抗原表达的影响机制的研究 　　[摘要]该文采用MTT法测定丹参素对转染人肝癌细胞株（HepG2）的2215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（HBsAg）和E抗原（HBeAg）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HepG2215细胞的生长起到良好的抑制作用，半抑制浓度IC50为（1535±243） μmol?L-1；丹参素能显著抑制HBsAg和HBeAg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（2132±243）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。 　　[关键词]丹参素；HepG2215细胞；乙肝病毒逆转录酶；活性氧；二级结构；抑制作用 　　[Abstract]MTT assay was used in this study to investigate the inhibitory effect of danshensu on the activity of 2215 cells among human hepatoma cell line （HepG2）； indirect fluorescence labeling method was used to measure the changes of reactive oxygen levels in the cells； ELISA method was used to determine hepatitis B surface antigen （HBsAg） and hepatitis B e antigen （HBeAg） levels in cellular supernatants； HBV DNA level was measured with fluorogenic quantitative PCR method The inhibitory effect of danshensu on HBV RT（hepatitis B virus reverse transcriptase） was studied by using enzyme inhibition dynamics， and the effect of danshensu on secondary structure of HBV reverse transcriptase was monitored by using circular dichroism The results showed that danshensu had a good inhibitory effect on the growth of HepG2215 cells， with a half inhibitory concentration （IC50） of （1535±243） μmol?L-1； danshensu could significantly inhibit HBsAg and HBeAg expressions， and showed an inhibitory effect on HBV DNA replication In addition， danshensu was an effective inhibitor for HBV reverse transcriptase [IC50 （2132±243） μmol?L-1] The fluorescence labeling results showed that the reactive oxygen levels in the cells were increased with the increase of danshensu concentration Circular dichroism analysis showed that danshensu could induce partial change of conformation of HBV reverse