低分子肝素体外抗人巨细胞病毒的作用

Inhibitory Effect of Low Molecular Weight Heparin on Human Cytomegalovirus in Vitro

目的探讨低分子肝素(LMWH)体外对人巨细胞病毒(HCMV)的抑制作用。方法根据病毒复制周期,设计3种抗病毒作用方式观察LMWH对HCMV的抑制作用。并设更昔洛韦(GCV)为阳性对照药。1.增殖抑制法:将人胚成纤维细胞(HF细胞)以病毒悬液吸附1.5 h后,弃去病毒液后加入含不同浓度LMWH。2.感染阻断法:HF细胞加不同浓度LMWH培养1.5 h,再加入100倍半数组织培养感染量(100TCID50)的病毒感染细胞后用细胞维持液培养。3.直接杀灭法:LMWH药物与病毒共同置于培养箱中1.5 h,再感染HF细胞,1.5 h后更换成细胞维持液培养。各实验组细胞置37℃、50 mL.L-1二氧化碳恒温箱内培养,逐日在显微镜下观察细胞病变结果。待病毒对照组出现明显病变时,采用四甲基偶氮唑盐(MTT)比色法测定A490值。计算细胞存活率和病毒抑制率,并使用Probit回归法计算2种药物对HCMV的半数抑制浓度(IC50)。结果 MTT法测定正常细胞对照组及病毒对照组A490值分别为0.386±0.024和0.045±0.002。在增殖抑制法中,GCV的IC50、治疗指数(TI)分别为352.6 mg.L-1和47.8。在感染阻断法和直接杀灭法中,LMWH的IC50、TI分别为2 016.9 mg.L-1、1.12和555.5 mg.L-1、4.07。结论 LMWH具有抗HCMV作用,但作用机制与GCV不同,其通过感染阻断和直接杀灭机制发挥抗HCMV的作用,具有较明显的量效关系。

Objective To explore the inhibitory effect of low molecular weight heparin（LMWH）on human cytomegalovirus（HCMV） in vitro.Methods According to viral replication cycle,3 anti-viral methods were designed to observe anti-HCMV effects of LMWH.With ganciclovir（GCV） as positive control.These anti-viral methods as following,1.Inhibiting virus multiplication：confluent cell monolayers were infected with HCMV.After adsorption at 37 ℃ for 1.5 h,it was replaced with the medium containing different served drug.2.Interrupting cells infection：HF cells were treated with the drug for 1.5 h.100 times media tissue culture infective dose of virus were added to HF cells with 0.1 mL per well and subsequently reincubated in drug-free medium.3.Directly killing virus：HCMV were incubated in LMWH or GCV medium at 37 ℃ for 1.5 h.Virus-drug mixed medium was added to HF cells 0.2 mL per well.After adsorption at 37 ℃ for 1.5 h,it was replaced by drug-free medium.All cells were cultured continuously at 37 ℃ in a humidified atmosphere of 50 mL·L^-1 CO2 and cytopathic effect（CPE） was observed under a microscope day by day.When the degree number of CPE was clear,the values of absorbency at 490 nm of all cell wells were measured by methyl thiazoyl tetrazolium colorimetry.The cell survival rate and drug inhibitory rate for HCMV were calculated.The final adoption of probit regression,the value of the median inhibitory concentration（IC50） of 2 drugs on HCMV were calculated,respectively.Results Measured by MTT colorimetry,the mean value of A490 of the virus controlled groups and normal HF cells groups were 0.045±0.002 and 0.386±0.024,respectively.Under drug addition with inhibiting virus multiplication method,the IC50 and treatment index（TI） of GCV group were 352.6 mg·L^-1 and 47.8,respectively.The IC50 and TI of LMWH were 2 016.9 mg·L^-1,1.12,and 555.5 mg·L^-1,4.07 by drug addition interrupting cells infection method and directly killing virus method,respectively.Conclusions LMWH has inhi-bitory effect on HCMV.Compared with GCV,LMWH has diffe-rent antiviral mechanisms of interrupting cells infection and directly killing virus in vitro,and showing clear dose-effect connection.