| Immune function is divided into natural immune and acquired immune. When two kinds of immune exercise their own function during various infections from pathogens, such as bacteria, parasite and virus, cytokines play important protective effects. Under normal circumstance, the infection from virus and bacteria first trigger natural immune, then induce the activation of Thl lymphocyte which is thought to be induced by IL-12 in the manner of antigen specificity. Infection with bacteria stimulates macrophage and subsequent NK cells to produce IL-12 and IFN- Y ,respectively.IL-12 enables NK cell to secret IFN- y which in turn promotes macrophage to present Ag to Ag-specific T cells. At last, natural immune and accompanying Ag-specific T cell response wipe out the pathogen together. So, IL-12 plays an important role against infection ,and is a key molecule to connect natural immune with acquired immune. However, the expression of IL-12 gene was suppressed during some infection, such as measles and leishmania infection. It was also reported that the erythrocyte infected with plasmodium berghei suppressed the function of dendritic cells and leaded the failure of type-I T lymphocyte response. In previous study, we ever observed the phenomenon that the expression of IL-12p40 gene of peritoneal exudes cells infected with P.berghei was suppressed, the phenomenon drove us to analyze the suppressed mechanism of IL-12p40 gene expression in P.berghei infection. Understanding the regulation on the expression of IL-12p40 gene is favorable for utilizing the function of IL-12 gene during infection, and preventing and controlling various infectious diseases.Part I :The gene expression of IL-12p40 of PEC from earlyphase infected mice with P.bergheiPlasmodium berghei passaged in mice was used as pathogen, and infected C57BL/6 mice 1 day by i.p. Collected peritoneal exudes cells (PEC), and measured the expression of IL-12p40 gene from uninfected and P.berghei-infected PEC of mice by RT-PCR. The results demonstrated that the mRNA expression of IL-12p40 gene from infected mice was profoundly lower than that from uninfected mice.To directly compare the amount of PCR products of a purpose gene in different experiments, we draw a standard curve for a gene of interest. The graded dose of IL-12p40 and DNA fragment (or plasmid such as pBluescript containing IL012p40 and 3 -actin target fragment) were used as templates, and the PCR products were visualized in agarose gel containing etidium bromide and recorded, and the intensity of the band were determined by NIH Image software 0 The results were plotted on semilog graph paper; the intensity (y axis) on a linear scale, and the amount of template (x axis) on a log scale. Both 3 -actin and IL-12p40 gave a straight line. Thus we could select 3 -actin as comparing standard for various molecules, i.e semiquantitative RT-PCR. We measured the amount of mRNA of IL-12p40 and IL-12p35 from uninfected and P.berghei infected mice. The results showed that P.berghei infection didn't influence the mRNA expression of IL-12p35, demonstrating the gene expression of IL-12p40 and IL-12p35 was separately regulated.Part II: The study of mechanism of IL-12p40 gene suppression from PEC ofplasmodium berghei-infected mice (1)------From the level of PECTo analyze the suppressed mechanism of IL-12p40 gene expression from P.berghei infected PEC cells, PEC cells were prepared 1 day after P.berghei-'mfected erythrocytes injection by i.p, stained with regular Giemsa and observed under photomicroscopy. The result showed that small proportion of PEC cells carried the pasitized erythrocytes in thesmall proportion of PEC cells carried the pasitized erythrocytes in the cytoplasm.We used cell fraction test. PEC cells of uninfected and P. berghei-infected mice were mixed according to different ratio, and were cultured on tissue culture plated for 24hs.Adherent cells were further cultured in the presence of LPS plus IFN- ,then measured the mRNA expression of IL-12p40 gene. The result revealed that R... |
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