| Objective:To explore the dynamic of complement mRNA from mouse macrophage ANA-1 infected with DENV-2 virus and its polarization. Methods: DENV-2 replicated in C6/36 cells was identified by RT-PCR and detected by TCID50. The polarized models of macrophage ANA-1 were established with LPS or IL-4 After treated 3h,8h,12h and 24h, macrophage polarization were estimated by detecting the expression of IL-1β, CCL2 and Arg-1 mRNA with Real-time fluorescent quantitative PCR. The intracellular complement C1q, C3, CfB and CRIg mRNA were quantitatively analyzed. Identify the infection of DENV-2 by PCR and the expression of NS1, Mavs and complement components mRNA were detected by Real-time PCR in M0/M2 polarized macrophage were infected by DENV-2. Results:1) The expression of complement mRNA in M1/M2 polarized ANA-1 cells:The intracellular complement C1q, C3, CfB and CRIg mRNAs all were up-regulated in M1/M2 polarized macrophage. ①The intracellular Clq and C3 mRNA in polarized M2 were significantly higher, in which the peak value of C1q and C3 were to 94.9±12.9 and 11.3±2.4 at 12h, respectively (P<0.05). ②The CfB mRNA in polarized M1 increased obviously, in which its 2-ΔΔct was to 61.4±6.2 at 12h. ③ The CRIg mRNA in both groups was only up-regulated at 24h, in which the 2-ΔΔct value was 6.5±1.8 in M1 and 10.8±3.2 in M2 (P<0.05).2) The expression of complement mRNA in M0/M2 polarized ANA-1 cells infected by DENV-2:Comparing with M2 uninfected group, the expression of C1q and C3 were reduced in M2 infected cells except for CRIg: ①The peak expression of C1q was 3.84±1.9 at 12h in M2 infected group; ②The highest expression of C3 was 5.08±2.25 in 12h. ③ The expression of CRIg in M2 infected group was induced. However in contrast with MO infected group:① The expression of C1q and CRIg mRNA was higher in M2 infected cell.② The expression of C3 mRNA was higher than MO infected group at 12h, thereafter it was opposite at 24h. ④ The mRNA of CfB was inhibited in ANA-1 infected by DENV-2, however there was no significant difference between MO and M2 polarization ANA-1.3) The dynamic characteristic of polarization in ANA-1 infected by DENV-2:The most expression of IL-1β, CCL2 and Arg-1 mRNA were inhibited in MO infected ANA-1 cells. The M2 polarization was delayed or reversed in ANA-1 cell infected by DENV-2.4) The expression of NS1, Mavs and complement components mRNA:There was no significant difference of the expression of NS1 and Mavs mRNA between M0 and M2 polarized ANA-1 cells infected by DENV-2. Conclusion:1) There are differences in the expression of complement mRNA between M1 and M2 polarized ANA-1 cells:The mRNA of CfB was higher in M1 polarization. However the mRNA of C1q, C3 and CRIg were higher in M2 polarization ANA-1.2) There are differences in the expression of complement mRNA in M0/M2 polarized ANA-1 infected by DENV-2:The expression of C1q and C3 were down-regulated in M2 infected ANA-1, while the mRNA of CRIg was in the regular or induced levels. The expression of C1q and CRIg were increased relatively comparing with M0 infected ANA-1.3) The polarization state was delayed or reversed by the infection of DENV.4) There was no significant relationship among the expression of complement, the multiplication of DENV-2 and anti-virus of ANA-1. |
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