| Objective: Through inoculated the mid-high titer serum of HBV virus,hepatitis B virus infected model were established in the newborn tree shrews and we investigate the immune reaction by detecting the dynamic changes of TLR-4and IL-6 subsequently.Methods:Part one: Through inoculated the mid-high titer serum of HBV virus,hepatitis B virus infected model were established in the newborn tree shrews.The new tree shrews were inoculated with the mid-titer serum(104-106IU/ml)and the high titer serum(107IU/ml)randomly and six new tree were not inoculated hepatitis B virus.From eighth weeks after inoculation,blood was collected through femoral artery every 2 weeks.After 12 weeks,liver biopsy was performed regularly every three month.The HBs Ag in serum was detected by ELISA,and the content of HBVDNA in liver tissue and serum was detected by fluorescence quantitative PCR(probe method).The expression of HBs Ag in liver tissues was observed by immunohistochemistry.Part two:Observing the dynamic changes of TLR-4 and IL-6 levels in serum and liver tissues in HBV infected animal model.the infection group was determined by persistent positive results of HBs Ag or HBVDNA.The 6 tree shrews withoutinoculated with human hepatitis B virus(HBV)were determined as control group.The levels of TLR-4 and IL-6 in peripheral blood and liver tissues were measured by enzyme-linked immunosorbent assay(ELISA)and HE staining separatly.The m RNA level of TLR-4 and IL-6 in the liver tissue of the infection group and the control group were detected by real-time fluorescence quantitative PCR.Results:Part one:A total of 34 newborn tree shrews were inoculated with human HBV virus,among 17 animals were inoculated with high titer group(7.53×107 IU/ml)and 17 animals were medium titer group(104?105?106IU/ml).(1)The results of serum HBs Ag: in the observation period(8 weeks and 24weeks),8 animals were found to be HBs Ag positive in the high titer group(8/17,47.05%).The HBs Ag in the middle titer group and the control group were negative.Among the 8 animals,7 were able to detect the HBs Ag as positive in eighth weeks and 1 for the first time in 22 weeks,and 5 of them were positive during the entire observation period(8~24 weeks),and the longest positive time lasted to 48 weeks.(2)Serum HBVDNA results:According to clinical standards,we defined HBVDNA?500IU/ml as positive.During the observation period(8~24 weeks),in the high titer group,11 HBVDNA positive sera were detected in.5 animals in the mid titer group could detect HBVDNA,but all were lower than the standard,while HBVDNA in the control group was negative.Most of the positive results in animals can be detected in eighth weeks,the shortest duration is 14 weeks and the longest is 72 weeks.The HBVDNA level of 11 positive animals was mostly around 103IU/ml,a few could reach104IU/ml and HBVDNA load gradually decreased with time.(3)HBVDNA results of liver tissue: 12 animals in the high titer group had HBVDNA positive(12/17,70.5%)in 12 weeks and 24 weeks of liver tissue examination;5 onlydetected HBVDNA positive(5/17,29.4%)in 12 th weeks in the middle titer group and 3 only detected HBVDNA positive(3/17,17.6%)in 24 weeks.No HBVDNA positive in the control group was detected in the liver tissue.In high titer group,the level of HBVDNA was more than 103 ~ 104IU/ml and a few could reach 105IU/ml.The HBVDNA level of positive animals in middle titer group was about 103IU/ml.(4)The results of immunohistochemistry showed that most of the animals in the high titer group and the middle titer group had the expression of HBs Ag and the HBs Ag positive animals in the high titer group were 12(12/17,70.5%),and the HBs Ag positive animals in the middle titer group were 5(5/17,29.4%).The control group had a negative result.The positive staining was brown and it expressed mainly in the cytoplasm and cell membrane of liver while.Part two:In the first part of the experiment,12 tree shrews with HBs Ag and / or HBV DNA positive were identified as the control group and Six uninoculated tree shrews with negative HBs Ag and HBVDNA were used as control group.The results of TLR-4 and IL-6 in both groups are as follows:(1)Among the infected group,The ELISA results of TLR-4 in infected group and control group showed that the TLR-4 concentration in serum of the infected group was higher than that of the control group in 8W,12 W,16W,20 W and 24W(P < 0.05),and the TLR-4 level decreased with the time.The serum concentration of IL-6 in the infection group was higher in 8W,12 W,16W,20 W and 24 W than the control group.The serum IL-6 level decreased slightly with time,and the increase of IL-6 in the infection group was different in 8W,12 W and 16 W compared with the control group(P < 0.05).(2)The m RNA expression level of TLR-4 and IL-6 in liver tissue of infected group was higher than that of control group by q RT-PCR,and there was difference between them(P < 0.05).(3)The expression rate of TLR-4 in the infection group was(10/12,83.3%)andthe control group was(1/6,16.6%)and the difference between the infection group and the control group was statistically significant(P<0.05).The expression rate of IL-6 in the infected group was(9/12,75%)and the control group was(1/6,16.6%)and the difference between the infection group and the control group was statistically significant(P < 0.05).Conclutions:1.Human HBV serum with high titer of 107 IU/ml could increase the positive rate of HBs Ag and HBVDNA in tree shrew serum,and prolong the duration of HBs Ag and HBVDNA positive in some extent,Compared with medium titer HBV serum,it is more advantageous to establish the animal model of hepatitis B in tree shrews.2.The expression of TLR-4 and IL-6 in serum and liver tissue of tree shrews infected with HBV was higher than that of normal tree shrews.Suggest that innate immunity and specific immunity play a role in the process of tree shrew infection with HBV,and TLR-4and IL-6 play an immune role in the process of HBV infection in tree shrews. |
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