| OBJECTIVE To establish a model of chronic hepatitis B infection in perinatal tree shrews by inoculating different titers of human HBV serum,to study the immune mechanism by monitoring the expression of cytokines IL-10 and IL-18 in vivo after inoculating serum HBV and feasibility of optimizing the model of perinatal Tupaia infected by HBV by regulating the immune link.METHODS Part One : Establishment of perinatal Tupaia was inoculated with mebium and high titer of HBV serum to optimize the chronic infection of Tupaia with HBV model.The newborn healthy tree shrews were randomly divided into three groups: the middle titer group(HBV DNA10~4 ~ 10~6 copies/ml)and the high titer group(HBV DNA10~7 ~ 10~8 copies/ml),6 were not vaccinated as control group.The HBV markers and HBV DNA(serum and livers tissue)was detected by ECLIA and PCR respectively after 8th weeks regulately.The expression of HBs Ag and HBc Ag in liver tissue were detected by immunohistochemistry.The pathological changes of liver tissue were observed by HE staining.Part Two: Observes the changes of IL-10 and IL-18 in the higher efficiency group.In this group,HBsAg positive and/or HBV DNA-positive tree shrews were included in the infection group and six were not inoculated as control group.The changes of IL-10 and IL-18 in the infection group were compared with those in the control group:(1)Test the levels of serum IL-10 and IL-18 at 8,12,16,20,24 weeks by ELISA;(2)The expression of IL-10 and IL-18 mRNA in liver tissue was detected by RT-PCR.(3)Observes the IL-10 and IL-18 in liver tissue by immunohistochemistry.RESULTS: Part One In the 36-week observation period,the moderate and high titers showed different infection efficiencies.The results were as follows:(1)serum ECLIA results: 8(50%)tree shrews were detected HBsAg positive since the 8th week,and 1(6.25%)HBeAg positive at the same time,2(12.5%)with HBeAb positive simultaneously.Most of them can be detceted HBsAg positive at 8th week,one tree shrew can be detected at 10 th week,most of them turn to negative after 16 th week.In the high titer group,5(31.25%)can be detected HBsAg positive,and 4(25%)with HBeAg positive and 1(6.25%)with HBsAb and HBeAb positive at the same time.Most can be detected HBs Ag positive at 8th week,one tree shrew can be detected HBs Ag positive at 14 th week,HBsAg positive duration ranging from 14 th week to 36 th week.(2)serum HBV DNA results: The serum HBV DNA of moderate titer group were negative.In the high titer group,10(62.5%)tree shrews can be detected HBV DNA positive after 8th week,and most of them maintained at 10~2 ~ 10~3 copies/ml,some reaching 10~4 copies/ml,up to 1.45 × 10~6 copies/ml;HBV DNA positive duration ranging from the shortest duration of 4 weeks after the first 16 weeks of inoculation disappeared,the longest duration can reach at 36 th week.With the prolongation of infection time,the virus titer showed a downward trend.(3)Detection of HBV DNA in liver tissue: liver biopsy was performed at the 20 th week and the 32 th week respectively.The positive rate of HBV DNA in the liver tissue of high titer group was higher than that in the middle titer group.The positive rate of HBV DNA at the 20 th week in high titer group(6,37.5%)is higher than that in the middle titer group(1,6.25%)(?~2 = 2.926,P = 0.083).The positive rate of HBV DNA at the 32 th week in the high titer group(9,56.25%)was significantly higher than that in the middle group(2,12.5%),(P<0.05).(4)The positive rate of HBsAg and HbcAg in liver tissue was 62.5% and 56.3% respectively in the high titer group,and the positive rates of HBsAg and HBcAg in the middle titer group were 43.7% and 37.5% respectively(P<0.05),but there was no significant difference between the two groups(P>0.05).The pathological changes of liver tissue were mainly hepatocyte swelling,cell infiltration changes.Part Two In the first part of the experiment,the infection efficiency of the animals in the high titer group was higher than that in the middle titer group,and the HBV infection maintained longer than the middle titer group.Therefore,We continue to observe the dynamic changes of IL-10 and IL-18 of this group.IL-10 and IL-18 levels in 11 HBsAg and/or HBV DNA-positive animals were compared with control group:(1)Dyna mic changes of serum IL-10 and IL-18 levels detected by ELISA:The level of periheral blood IL-10 and IL-18 of infention group were higher than that in the control group at 8,12,16,20,24 th week.Compared with the control group,the increase of IL-18 was significant difference(P<0.05).And the concentration of IL-10 in peripheral blood was significantly higher than that in control group at 16,20,24 th week(P<0.05).(2)The expression of IL-10 m RNA in liver tissue was significantly higher than that in control group(P<0.05).While the expression of IL-18 m RNA increased,but the difference was not significant(P>0.05).(3)The positive rate of IL-10 protein expression in the liver tissue of the infected group was 81.8%,which was significantly higher than that in the control group(16.6%)(P<0.05).The positive rate of IL-18 protein rate was 72.7%,compared with the control group(16.6%),the difference was statistically significant(P?0.05).Conclusions: 1.The selection of 10~7 ~ 10~8 copies/ml HBV serum is more beneficial to the replication of human HBV in the newborn tree shrew,and is more beneficia to prolong the infection time than the inoculation of 10~4 ~ 10~6 copies/ml,and improving the positive rate of HBsAg may be beneficial to optimize the model of chronic hepatitis B infection in tree shrew.2.After infecting HBV,the levels of IL-10 and IL-18 in serum and liver tissue were higher than those in normal tree shrews.These results indicated that the adaptive immune response was activated after infecting HBV,and IL-10 and IL-18 may be play an important role in the clearance of HBV and have a significant impact on viral infection progress. |
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