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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 investigate the inhibitory effect of low molecular weight heparin (LMWH) on human cytomegalovirus (HCMV) in vitro. Methods According to the virus replication cycle, three kinds of antiviral effects were designed to observe the inhibitory effect of LMWH on HCMV. And set ganciclovir (GCV) as a positive control drug. 1. Proliferation inhibition: human embryonic fibroblasts (HF cells) to the virus suspension adsorption 1.5 h, the virus was discarded after adding different concentrations of LMWH. 2. Infection blocking method: HF cells with different concentrations of LMWH cultured 1.5 h, then add 100 times half of the tissue culture infection (100TCID50) virus infected cells cultured with cell maintenance fluid. 3. Direct kill method: LMWH drugs and viruses were placed in an incubator for 1.5 h, and then infected with HF cells, replaced after 1.5 h cell maintenance fluid culture. Each experimental group cells were incubated in a 37 ° C, 50 mL.L-1 carbon dioxide incubator, and the cytopathic effect was observed daily under the microscope. When obvious lesions were found in the virus control group, A490 values were determined by MTT colorimetry. Cell viability and virus inhibition rates were calculated and half-maximal inhibitory concentration (IC50) of two drugs on HCMV was calculated using Probit regression. Results The A490 values of normal control group and virus control group measured by MTT assay were 0.386 ± 0.024 and 0.045 ± 0.002, respectively. In the proliferation inhibition assay, the IC50 and the therapeutic index (TI) of GCV 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 respectively in the infection-blocking and direct-killing methods. Conclusion LMWH has an anti-HCMV effect, but its mechanism is different from that of GCV. It exerts an anti-HCMV effect by blocking infection and direct killing mechanism and has a significant dose-response relationship.