The Inhibitory Effect Of Low Molecular Weight Heparin On Human Cytomegalovirus In Vitro

Objective:To study the inhibitory effect of Low Molecular Weight Heparin (LMWH) on human cytomegalovirus (HCMV) in vitro.Methods:1. The cytotoxic effects of Human Cytomegalovirus in Human embryo fibroblast (HF) cells were examined by Cytopathic effect (CPE). Stocks of HCMV AD 169 strains were diluted by Dulbecco's Modified Eagles Medium (DMEM) from 10-1 to 10-8. There were served in 8 wells every concentration. Confluent cell monolayers in 96-well Plates were infected with different concentration virus, and normal HF cells served as negative controlled group. Cell cultures were incubated at 37℃in a humidified atmosphere of 5%CO2-95%air and cytopathic effects (CPE) were observed under a microscope day by day. The degree number of CPE was enregistered until cytopathic effects did not aggravate. Tissue cell infection median dose(TCID50) were calculated by Reed-Muench method. Pathological changes of HF cells exceeded 50% were determinanted as positive well.2. The cytotoxic effects of LMWH in HF cells were examined by MTT colorimetry. LMWH of seven different concentrations were added in monolayers of HF cells in 96-well Plates. There were served 8 wells every concentration. Ganciclovir group and normal HF cells served concurrently as positive controlled group and negative controlled group, respectively. Cell cultures were incubated at 37℃in a humidified atmosphere of 5%CO2-95% air. The cytopathic effects of HF cells by two kinds of drug were observed persistently under a microscope for a week until cytopathic effects did not aggravate. The values of absorbency at 490nm(A490) of all cell wells were measured by MTT colorimetry. Median cytotoxic concentration (TC50) were calculated by Probit regression method.3. According to viral replication cycle, three anti-viral methods were designed to observe anti-HCMV effects of LMWH. Stocks of HCMV AD169 strains were diluted by DMEM to 100 times of TCID50. The different drug LMWH addition methods was researched by MTT colorimetry, and Ganciclovir was served as positive reference drug group. Untreated HF cells and infected HF cells were served as normal cell control group and virus control group, respectively. Three anti-viral methods as following:Method 1, inhibiting virus multiplication, Confluent cell monolayers in 96-well plates were infected with HCMV. After adsorption at 37℃for 2h, it was replaced with the medium containing different served drug. Method 2, interrupting cells infection, HF cells were treated with two kinds of drug for 2h, washed twice with PBS.100TCID50 of virus were added to HF cells with 0.1ml per well and subsequently reincubated in drug-free medium. Method 3, directly killing virus, HCMV were incubated in LMWH or GCV medium at 37℃for 2h. Virus-drug mixed medium was added to HF cells 0.2ml per well. After adsorption at 37℃for 2h, it was replaced by drug-free medium. All cells were cultured continuously at 37℃in a humidified atmosphere of 5%CO2-95%air and CPE were observed under a microscope day by day. When the degree number of CPE was clear, the values of absorbency at 490nm of all cell wells were measured by MTT colorimetry. The cell survival rate (CSR) and drug inhibitory rate(IR) for HCMV were calculated. By Probit regression method, the Median inhibitory concentration (IC50) of two drugs were calculated respectively.Result:1. The results shew that the TCID50 Of HCMV was 10-4.67/0.1 ml after stocks of HCMV AD 169 strains were diluted from 10-1to 10 -8.2. The results shew that the TC0 and the TC50 of LMWH were 2500 and 2261.7 mg/L by MTT colorimetry, respectively. Corresponding values of GCV were 800 and 1685.23mg/L, respectively.3. Three anti-viral methods were designed to observe anti-HCMV effects. By drug addition method 1, the CPE were restrained clearly in GCV group. The CPE were only inhibited at different degrees in LMWH group under drug addition method 2 and 3. By drug addition method 1, compared with the values of A490 of virus controlled groups, the values of A490 of GCV in all concentrations were statistically significant (P<0.05). The values of GCV group were not statistically differences (P>0.05) under drug addition method 2 and 3. Corresponding values of LMWH group were not statistically differences (P>0.05) under drug addition method 1. By drug addition method 2 and 3, The values of LMWH group were almost statistically significant in all concentrations. (P<0.05 or P<0.01). By different drug addition methods, the CPE in HF cells were observed in all parallel virus controlled groups, while normal cell control group were absolute negative.4. Measured by MTT colorimetry, the mean value of A490 of the Virus controlled groups and normal HF cells groups were 0.045±0.002,0.386±0.024, respectively. Under drug addition method 1, the median inhibitory concentration (IC₅₀) and Treatment index (TI) of GCV group were 35.26mg/L and 47.8, respectively. The IC₅₀ and TI of LMWH were 2016.9 mg/L,1.12 and 555.46 mg/L,4.07 by drug addition method 2 and 3, respectively.5. The charts of dose-effect by different drug addition methods were recorded, serving drug concentrations and IR as abscissa and ordinate respectively. Under drug addition method 1, the inhibitory effects of GCV became stronger with the increase of concentration, showing clear dose-effect connection. Under drug addition method 2 and 3, the results shew partial dose-effect connection.Conclusion:1. According to the result in vitro, it shew clear inhibitory effect of LMWH on HCMV. The effects became stronger with the increase of concentration, showing partial dose-effect connection. 2.LMWH group didn't show clear dose-effect connection in inhibiting multiplication of HCMV and directly killing virus. But LMWH had an activity for guarding against HF cells'infection and restraining infection by HCMV at several effect points in vitro.