| Objective To optimize the tree shrew model chronically infected by human HBV by inoculating newborn tree shrew with low copy number of human serum HBV; to study immune response mechanisms in the host of HBV infection by monitoring the expression of immune-related factors IFN-y and TNF-a in vivo after inoculating human HBV and feasibility of optimizing the model of tree shrew infected by HBV by regulating the immune link.Methods Part one: Establishment of newborn tree shrew model infected by human HBV, the main contents are: newborn tree shrew inoculated by human HBV serum with low copy number (103copies/ml) through bilateral thigh subcutaneously, dynamically monitoring markers of HBV infection in serum and hepatic tissue with ELISA and PCR after 8 weeks, while observing pathological changes of the tree shrews' liver tissue. Part two : Analysis of levels of immune-related factor IFN-y and TNF-a in vivo of tree shrews after HBV infection, the main contents are: infection group including 18 tree shrews with HBsAg and / or HBV DNA positive, control group including six normal tree shrews without HBV inoculation. Test the serum and liver tissues of all animals by following: ? mRNA expression levels of immune-related factor IFN-y and TNF-a in liver tissue by real-time fluorescent quantitative RT-PCR (qRT-PCR);? expression of IFN-? and TNF-? in liver tissue by immunohistochemistry;?IFN-y and TNF-a changes levels in peripheral blood 8,12,16,20,24 and 28 weeks after HBV inoculation by ELISA.Results Part one : Results after inoculation of 28 newborn tree shrews during observation period are as follows:? serum ELISA results showed that HBsAg positive (28/28,100%) in serum samples were successively detected in the 8th week after inoculation, and showed continued fluctuation, with the duration up to 22 weeks, HBsAg became negative in most of the animals after the 20th week; where in 8 (28.6%) are HBsAb positive in different time, which was a transient performance; 4 (14.3%) detected HBeAg-positive; 5 (17.9%)detected HBeAb positive; 2 (7.1%) detected HBcAb positive. ? serum or liver tissue PCR results showed that since the eighth week after inoculation HBV DNA viral copy number could be detected in serum, which is the peak, and thereafter, the number dropped, until 24 weeks after vaccination, HBV DNA were undetectable in the serum of all animals; 9 (32.1%) samples were detected HBV DNA in liver tissue, and showed low levels (101?103/ug the liver total DNA), only one was detected HBV DNA in both serum and liver tissue. ?Expression of HBcAg and main pathological changes in animal liver tissues are as follows: HBcAg positive cells are expressed in most (60.7%, 17/28) animal liver tissues; most can be seen liver tissue swelling, cytoplasm lightly stained,watery degeneration, a small amount of monocytes or lymphocytes and other inflammatory cell infiltration in hepatic lobular, and several spotty necrosis;small portion of liver tissue did not exhibit significant pathological and histological changes.Part two: Level of immune-related cytokines in infection group and control group:? qRT-PCR test results showed that IFN-y mRNA expression level was higher in infection group (0.291 ±0.098) than control group(0.096±0.046), there is significant statistically difference (P<0.05) between the two groups; TNF-? mRNA expression level was higher in infection group(0.542±0.163) than control group (0.411 ±0.096), but the difference between the two groups is not statistically significant (P>0.05).? Immunohistochemistry results showed that the positive rate of IFN-y protein in liver tissue of the two groups were 66.7%(12/18) and 0% (0/6), the difference is statistically significant(P <0.05) ; the positive rate of TNF-a protein in two groups were 88.9% (16/18)and 16.7% (1/6) respectively, the difference was statistically significant (P<0.05).? ELISA dynamic observation of IFN-y and TNF-a level in peripheral blood showed that each time IFN-y and TNF-a in two groups were detected at low levels, which were under the detection threshold of the kit.Conclusion 1. Human serum with HBV of low copy number (103copies /ml) can avoid being removed by the host immune response in vivo activated rapidly after inoculation, increasing detection rate of HBsAg in serum of tree shrews and extending time of HBsAb occurrence, and is expected to improve chronically HBV infection rate in tree shrews. 2. The tree shrews have humanlike seroconversion process and hepatic pathological changes in the course of HBV infection, which is an ideal animal model in related fields of human HBV infection. 3. Immune-related factors IFN-y, TNF-a expression level were much higher in HBV infected tree shrews than that of normal tree shrews,prompting that immune response of tree shrews after in trahepatic HBV infection was established by up regulating the expression of immune-related factors, thus plays a role in clearing the virus and virus infection process. Tree shrews may have a similar immune response to humans after HBV infection. |
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