Cytokines In C57BL/6 Mice Model Of Hepatitis B Virus Infection

Backgrounds and ObjectiveBy establishing the C57BL/6 mice model of HBV infection and considering the gene microarrays as the references, the differences in the expression of chemokines such as IL-18,Mig,IP-10,CXCL12,CCR7 and CCL19, as well as the partial genes for acute phase proteins and immune-associated proteins were detected to further study the function and mechanism of the inflammatory factors in HBV infection.Methods1. The establishment of C57BL/6 mice model of HBV infection: with the preparation of super-competent bacteria DH5α, pBluescript and pBluescript-HBV plasmids were extracted to the mice by the method of high pressure water via caudal vein injection. And the mice with pBluescript and pBluescript-HBV were assigned into control and experimental group respectively.2. In both groups the peripheral blood was sampled to test the levels of HBsAg at 4th day. And the tests were estimated the establishment of the animal model. Meanwhile the livers in both groups were cryopreserved.3. According to the specific cytokines expressed significantly different, the primers were designed and sequences were synthesized. By method of Trizol, the hepatic total RNA was extracted to synthesize the primers. The numbers of the objective gene copies were analyzed by Real-Time PCR. RT-PCR gel electrophoresis was used to analyze the synthetic primer, and density and gray scale of the electrophoretic bands were also scanned and evaluated.4. Cytokines expressed significantly different between the two groups, such as IP-10, were tested by immunohistochemistry.Results 1. The C57BL/6 mice model of HBV infection: the positive ELISA test of HBsAg suggested the animal model using high pressure water via caudal vein injection was successful.2. The expressions of chemokines in the lives of the C57BL/6 mice model of HBV infection:①in the Real-time PCR test, the amplification curve and melting curve performed favorable; and compared with the controls, the expression of IL-18,Mig,IP-10,Cxcl12,Ccl19 and Ccr7 in experimental group, especially IP-10 were increased by degrees. The numbers of gene copies of chemokines above, especially IP-10 were increased by degrees in RT-PCR test, and the results identified with the Real-Time PCR test.②According to the immunohistochemistry test, the expression of IP-10 compared with the controls was increased significantly.3. The expressions of immune-response molecular and acute phase proteins in the lives of the C57BL/6 mice model of HBV infection: in the Real-time PCR test, the amplification curve and melting curve performed favorable; and compared with the controls, the expression of acute phase response related factor Saa2 increased in experimental group. In the RT-PCT test, the expression of heat shock protein related gene Dnajc10, acute phase response related factor Saa2, inherent immune response related molecular Masp1, and anti-apoptosis related factor Vnn1 were increased by degrees in experimental arm.ConclusionsThe level of HBsAg in the ELISA test demonstrated the C57BL/6 mice model of HBV infection was established successfully. Compared with the controls, the expressions of chemokines IL-18, Mig, IP-10, Cxcl12, Ccl19 and Ccr7 all increased by degrees in the Real-time PCR and RT-PCR test; the expression of IP-10 increased significantly in both PCR and immunohistochemistry test. The results suggested the expression of the chemokines increased by degrees in HBV infection and further demonstrated acute phase and immune-response related molecular increased by degrees, but the function and mechanism of the high expression need further research.