| Objectives: To select experimental animal models suitable for establishment of HBV replication from mice of three different strains of Kunming,BALB/c and C57BL/6,preliminarily discuss the anti-HBV effect of ICSII in vivo;to study the anti-HBV effect of ICSII in vitro;and to explore the mechanism of ICSII anti-HBV.Methods: Part One: 1.Three different mice strains of Kunming,BALB/c and C57BL/6 were selected to establish the HBV replication mouse model by using the method of hydrodynamic tail vein injection of p AAV-HBV1.2 plasmid.The serum was collected by centrifugation,real time quantitative PCR technique was used to detect the copy number of HBV DNA in sera of different strain mice.When it is below or equal to the clinical positive standard,it is not being monitored.ELISA method was used to detect the expression of HBs Ag and HBe Ag in sera of different strains of mice.The duration of HBV DNA(positive expression)and the positive rate of HBs Ag and HBe Ag in the serum of different strains of mice were calculated,and a better mouse model suitable for HBV replication was selected.2.Based on the above results,the HBV replicated mouse model was selected as the research basis.They were divided into three groups: model control group,ICSII group and entecavir(entecavir,ENT)positive drug control group.They were given by gavage continuously with normal saline,ICSII(20mg/kg)and ENT(0.5mg/kg)for 20 days.The blood samples collected from the orbital veins on the 10 th and 20 th.Then,the expression of HBV DNA copies in serum samples of each group were detected by real time quantitative PCR technique,and the HBs Ag,HBe Ag in the serum samples of each group was detected by ELISA.Part Two: 1.To determine the toxic effects of different concentrations of ICSII on Hep G2.2.15 cells by CCK8.2.The effect of ICSII on the secretion of HBV DNA from Hep G2.2.15 cells was detected by real time quantitative PCR technique.3.The effect of ICSII on the secretion of HBs Ag and HBe Ag from Hep G2.2.15 cells was detected by ELISA.Part Three: 1.The effect of ICSII on the expression of GSH and GST in Hep G2.2.15 cells was detected by ELISA.2.The effect of ICSII on the SOD1 expression in Hep G2.2.15 cells was detected by Western-blot.Results: Part One: 1.HBV DNA: On the 4th day after tail vein injection of p AAV-HBV1.2 plasmid,the replication of HBV DNA was detected in the serum of Kunming mice,BALB/c and C57BL/6 mice,and the positive rate of transfection was 100%.Statistical analysis showed that the positive expression time of HBV DNA in C57BL/6 mice was higher than that in Kunming mice and BALB/c mice,and the difference was statistically significant.HBs Ag: The positive rate of HBs Ag in Kunming,BALB/c and C57BL/6 mice was nearly 20%,95% and 95%.Statistical analysis showed that the positive rate of HBs Ag in C57BL/6 mice and BALB/c mice was higher than Kunming mice,and the difference was statistically significant.HBe Ag: the positive rate of HBe Ag in Kunming mice was nearly 10%,BALB/c mice was nearly 20%,C57BL/6 mice was nearly 90%.Statistical analysis showed that the positive rate of HBe Ag in C57BL/6mice was higher than that in Kunming mice and BALB/c mice.The difference was statistically significant.2.The HBV-replicated C57BL/6 mice were used as an in vivo experimental model.After ICSII(20mg/kg)continuous intragastric administration of mice for 10 days and 20 days,the replication of HBV DNA and the expression of HBs Ag and HBe Ag in mice serum were all inhibited.In the administration of 10 d,the inhibition rate of ICSII on HBV DNA,HBs Ag,HBe Ag were 84.49%,13.92%,18.36%,compared with the model control group,the differences were statistically significant(P<0.05);At 20 d after administration,the inhibition rate of ICSII on HBV DNA,HBs Ag,HBe Ag were 65.38%,50.62%,43.05%,compared with the model control group,the differences were statistically significant(P<0.05).Part Two: 1.ICSII had no obvious toxic effect on Hep G2.2.15 cells in the concentration range of 12.5 ?g/m L.2.The low(6.25?g/m L),medium(10?g/m L)and high(12.5?g/m L)concentration group of ICSII had inhibitory effects on Hep G2.2.15 cells secreting HBV DNA in 24 h,48h,and 72 h.The inhibitory effect was gradually enhanced with the increase of drug concentration.Statistical analysis showed that: the medium concentration group of ICSII in 24 h,48h and high concentration group of ICSII in 24 h,48h,72 h,all had significant inhibitory effects on HBV DNA,compared with the control group,the differences were statistically significant(P<0.05).3.The low(6.25?g/m L),medium(10?g/m L),and high(12.5?g/m L)concentrations of ICSII in 24 h,48h,72 h inhibit the secretion of HBs Ag in Hep G2.2.15 cells.The inhibitory effect was gradually enhanced with the increase of drug concentration.Statistical analysis showed that: the medium(10?g/m L)and high(12.5?g/m L)concentration group of ICSII had significant inhibitory effects on HBs Ag and HBe Ag in 24 h,48h,72 h,compared with the control group,the differences were statistically significant(P<0.05).Part Three: 1.After ICSII(12.5?g/m L)was given Hep G2.2.15 cells 72 h,the expression of GSH and GST increased,and the difference was statistically significant compared with the control group(P<0.05).2.After ICSII(12.5?g/m L)was given Hep G2.2.15 cells 72 h,the expression of SOD1 increased,and the difference was statistically significant compared with the control group(P<0.05).Conclusions: Part One: 1.C57BL/6 mice are more suitable for the establishment of HBV replication mouse models.2.ICSII can inhibit the replication of HBV DNA and the expression of HBs Ag and HBe Ag in the serum of HBV-replicated C57BL/6 mice which is a potential anti-HBV active ingredient in Chinese medicine.Part Two: 1.ICSII had no obvious toxic effect on Hep G2.2.15 cells in the concentration range of 12.5?g/m L.2.ICSII has a significant inhibitory effect on the secretion of HBV DNA in Hep G2.2.15 cells.3.ICSII had a significant inhibitory effect on the secretion of HBs Ag and HBe Ag in Hep G2.2.15 cells.Part Three: Oxidative stress injury may be one of the mechanisms of ICSII anti-HBV. |
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