| Porcine reproductive and respiratory syndrome, PRRS is a new type of viral infection first discovered in the United States in 1987, which is now one of the most important disease that may harm the pig farming industry in the world. Macrophage is an immune system cell that has a wide range of distribution, which can be changed extremely into different types according to the changes of the internal environment: classical activated macrophages (M1), as well as alternative activated macrophages (M2). Porcine alveolar macrophage, PAMs is only one of the most important target cells in the pig body after being infected the PRRSV. Therefore, this study shall first study the effect of PAMs polarization on PRRSV infection and the dynamic change of the effect of PRRSV infection on PAMs through establishing the PAMs polarization in vitro model. This experiment used the method of pre-cooling to separate PAMs and cultured it in vitro, used LPS/IFN-γ respectively to induce and stimulate Ml macrophages, and used IL-4 to induce and stimulate M2 macrophages. Detect the change of the quantity of Ml-type, M2-type Marker (M1:IL-1β, IL-12, IL-23 and CXCL10; M2:Arg-1, IL-10, PPARγ and YM-1) respectively through the semi-quantitative PCR, and prove the success of the polarization model from the nucleic acid level. At the same time, use the Western blotting to determine the success of Ml-type and M2-type PAMs polarization from the protein levels. Afterwards, use the PAMs subtype after being infected and polarized by the 500TCID50 HuN4-F5 strains of PRRSV to detect the PRRSV infection by IFA and Western blotting after the hours of 12,24,36 and 48. Seen from the results, we found that within 12 to 36 hours after infection, the amount of PRRSV infection in M1 PAMs was significantly lower than that of the normal group and the type M2 PAMs, while at the 48 hours after infection, the amount of PRRSV infection in M1 PAMs was almost the same with that of the normal group and the type M2 PAMs, there was no significant difference.However, PAMs mainly plays its function of the innate immune defense through its pattern recognition receptor, PRR. So whether the PRRSV infection of M1 macrophages has an effect on the pattern recognition receptors, so as to make the M1 macrophages of PRRSV infection produce the inhibition over a period of time. So, by the method of fluorescence quantitative PCR, we carried on a detection to the expression of the recognition receptor of Ml macrophages pattern mRNA that has been infected by the PRRSV, and at the same time, we also detected the KLF4 gene that was considered to be key regulators of macrophage polarization. In the determination results, we accidentally found that after the PRRSV infection and polarization, the replication of KLF4 expression and M1 macrophages tends to have the similar effects on PRRSV. The results show that after the PRRSV infection, as the change of time, the KLF4 expression in Ml macrophages gradually reduces and reaches to the minimum 12 hours later, while the KLF4 expression quantity shall gradually increase after 24 hours, and the KLF4 expression quantity shall gradually be the same with that of the normal group after 48 hours, and there was no any significant difference.Therefore, in order to further study the influence of the pig KLF4 gene on PRRSV replication, this experiment obtained the KLF4 gene by PCR amplification, and built pMD18-T-KLF4 recombinant plasmid by TA cloning. After the detection of the enzyme digestion, input the cDNA cloning of KLF4 to pCMV-HA carrier, thus to construct the recombinant plasmid pCMV-HA-KLF4. The transfection recombinant plasmid pCMV-HA-KLF4 shall infect the PRRSV after 12 hours in Marc-145, and detect its quantity of PRRSV infection by Western blotting after 24 hours of the infection. The results show that KLF4 gene of PRRSV infection have a certain role in promoting. |
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