低分子质量肝素联合更昔洛韦体外抗人巨细胞病毒的作用

Inhibitive Effects of Low Molecular Weight Heparin Combined with Gancilovir on Human Cytomegalovirus in Vitro

目的研究低分子质量肝素(LMWH)联合更昔洛韦(GCV)体外抗人巨细胞病毒(HCMV)感染的作用。方法采用四甲基偶氮唑盐(MTT)比色法测定LMWH联合GCV对人胚肺成纤维细胞(HELF)的最大无毒质量浓度(TC0)。应用体外细胞培养技术建立HCMV-A;;DOI:10.3969/j.issn.169感染HELF模型。固定联合药物中LMWH的TC0(625.00 mg.L-1),设LWMH 625.00 mg.L-1、GCV 35.00mg.L-1、GCV 17.50 mg.L-1、GCV 8.75 mg.L-1、LMWH 625.00 mg.L-1+GCV 35.00 mg.L-1、LMWH 625.00 mg.L-1+GCV17.50 mg.L-1、LMWH 625.00 mg.L-1+GCV 8.75 mg.L-17个不同用药组,分别干预HCMV感染细胞模型,使用MTT法检测各组细胞存活率,观察感染细胞特征性细胞病变(CPE),同时应用荧光定量PCR法检测用药后各组HCMV的载量。结果联合药物的TC0中GCV的质量浓度为375.00 mg.L-1,LMWH的质量浓度为625.00 mg.L-1。当HCMV A;;DOI:10.3969/j.issn.169浓度为100倍半数组织培养感染量(TCI;;DOI:10.3969/j.issn.50)感染HELF时,LMWH和GCV均可提高受染细胞存活率,降低受染细胞的HCMV ;;DOI:10.3969/j.issn.NA的拷贝数;当固定LMWH的TC0,与3种不同质量浓度GCV配伍时,均在HELF上有明显的抗HCMV的生物活性,并显示配伍后的抑制作用明显大于对应质量浓度单用GCV的抑制作用(P<0.01);当联合用药中GCV剂量减半时与全量应用GCV时的抗HCMV效果相同[即GCV 35.00 mg.L-1组与联合用药LMWH 625.00 mg.L-1+GCV 17.50 mg.L-1组的病毒抑制率和;;DOI:10.3969/j.issn.NA拷贝数比较差异无统计学意义(P>0.05)];且联合用药对细胞的毒性试验结果及显微镜下观察细胞形态变化显示,随着联合用药中二者药物质量浓度的下降,细胞存活率升高,对细胞的毒性作用减弱。结论 LMWH联合GCV具有良好的体外抗HCMV作用,二者具有协同抗病毒作用。

Objective To study the inhibitive effect of low molecular weight heparin（LMWH） combined with gancilovir（GCV） on human cytomegalovirus（HCMV） infection in vitro.Methods HCMV infected cell model was established by coincubating the human embryonic lung fibroblast（HELF） and HCMV-AD169 with titer of 100 times median tissue culture infective dose（100TCID50） in vitro.The 0% toxic concentration（TC0） of LMWH and GCV were detected by using methyl thiazolyl tetrazolium（MTT） colorimetric method and TC0 of LMWH was fixed at 625.00 mg·L-1.It was categorized into 7 different groups： LWMH 625.00 mg·L-1,GCV 35.00 mg·L-1,GCV 17.50 mg·L-1,GCV 8.75 mg·L-1,LMWH 625.00 mg·L-1＋ GCV 35.00 mg·L-1,LMWH 625.00 mg·L-1＋GCV 17.50 mg·L-1,LMWH 625.00 mg·L-1＋GCV 8.75 mg·L-1.The cell survival rates were detected by using MTT method,and the development of cytopathic effect caused by HCMV was observed before and after being treated with drugs respectively.Meanwhile,the HCMV DNA copy number in cultured cell supernatant was determined by using fluorescence quantitative polymerase chain reaction.Results The TC0 of LMWH and GCV were 625.00 mg·L-1 and 375.00 mg·L-1 respectively,and both LMWH and GCV could increase the infected cells survival rates and decrease the HCMV DNA copy number in case that the HELF cell should get infected by HCMV-AD169 with titer of 100TCID50.When the fixed concentration of LMWH（625.00 mg·L-1） was combined with 3 different concentrations of GCV,it showed great anti-HCMV bioactivity and enhanced inhibitory effect in the combination groups than separate group（P0.01）.Also,it showed that there was no significant diffe-rence in virus inhibitory rates and DNA copy number（P0.05） in group GCV（35.00 mg·L-1） and group LMWH（625.00 mg·L-1）＋GCV（17.50 mg·L-1）.Moreover,it showed that the infected cell survival rates ascended and the toxic effects of the drug on the cell wea-kened with the drop of the concentration of the combination drugs according to the result of the combination drugs′ toxic effects on the cells and the cell morphology changes were observed under microscope.Conclusion The combination of LMWH and GCV shows obvious inhibitive effect on HCMV and they shows a synergetic antiviral effect in vitro.