| Summary: | 碩士 === 東吳大學 === 微生物學系 === 92 === Abstract
In crustacean, both prophenoloxidase activating system (PAS) and phagocytosis play important roles in the immune system and are believed to be used as the defense indicator. In shrimp, it is very difficult to determine the immune reactions of hemocytes since the different individuals are variable. Therefore, a cell line is necessary and important in the study of defense mechanism. The previous study showed that CpG ODN can direct stimulate the hemocytes via binding to the ODN receptor-like molecules on cell surface; thereafter, degranulation and PAS activity of stimulated hemocytes are triggered via either G protein/protein kinase C (PKC) or cAMP pathway (Chuo et al., 2004).
In this study, in order to study the influence of CpG ODN on the PAS activity and phagocytic activity of PMO cells, the PMO cell line, which was derived from Oka organ of Penaeus monodon and did not express the PAS, was first induced to be further differentiated by treatment with a cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). The result showed that neither PO activity nor proPO mRNA of GM-CSF-induced PMO cells was detectable, but phagocytic activity was detected after cells were treated with LPS, ODN2006 or so-ODN13.
In order to observe the localization of ODN2006 on or within PMO cells after stimulation by ODN, in this experiment, cells were directly stimulated and labeled with the FAM-labeled ODN and observed by a confocal microscope. To compare with the un-stimulated control sample, it was found that the fluorescent intensity of cells was gradually increased according to the increase of the reaction time. Following the change in the length of stimulating time, the ODN molecules were randomly located on cell membrane and within cytosol, polarized on one side of nucleus, and then polarized on both side of nucleus. After cells were continuously stimulated by ODN2006 for 15 min, the whole cell, including cell membrane and cytosol, contained a large amount of ODN. The phenomenon of ODN localization within PMO cells consistanted with that within haemocytes of Macrobrachium rosenbergii, even within the macrophages of mammalian.
In order to understand the properties of the ODN-labeled PMO cells, in this experiment, cells were directly stimulated and labeled with the FAM-labeled ODN and detected by a flow cytometer. The results showed that not noly the number of ODN-labeled cells increased, but also the fluorescent intensity of the whole cell increased; however, there were 10% of un-labeled cells. In addition, from 1 min to 10 min after stimulation, it was found that, in the group of the cytosol labeled by ODN, the percentage of cells with weak fluorescent intensity increased; meanwhile, in the group of cell membrane labeled by ODN, the percentage of cells with high fluorescent light also increased. It was speculated that, during this period, ODN seemed to move from the cytosol to the cell membrane. From 10 min to 30 min after stimulation, it was found that, in both the group of the cytosol labeled by ODN and the group of cell membrane labeled by ODN, the percentage of cells with high fluorescent light increased. The results were suggested that new ODN-binding proteins may be produced during this period.
To further characterize the ODN-binding proteins of PMO cells, using the procedure of ODN absorption, two proteins which may be ODN-binding molecules, were separated and purified. It was found that the molecular weights of the two proteins were respectively about 60 kDa and 65 kDa which are smaller than those of mammalian TLR9 with 115 kDa to 120 kDa. After their peptide sequence was analyzed by MALDI-TOF mass spectrometry, it was found that two proteins were not associated with TLR9. Furthermore, to determine the relationship of ODN-binding proteins presented in PMO cells and TLR9, in this study, ODN and anti-TLR9 antibody were used in a competitive test. The results showed that anti-TLR9 antibody was not able to compete against ODN to bind the ODN-binding site on the surface of PMO. It was speculated that the structureal similarity between ODN-binding protein and TLR9 may be low.
According to the previous study, this study primarily focused on studing the G protein/PKC signal pathway when phagocytic activity of PMO cells was enhanced by ODN. The results showed that the phagocytic activity of GM-CSF-treated PMO cells was increased by either NaF (G protein activator) or Phorbol-12-myristate-13-acetate (PMA; a PKC activator). The activity was slightly decreased after the cells were co-treated with PMA and chelerythrine (a PKC inhibitor); but not changed after cells co-treated with ODN2006 and chelerythrine. Besides, the relationship of the intracellular calcium concentration and the ODN-triggered signaling pathway was also determined. The results showed that neither in the ODN-treated PMO cells nor untreated cells, the change of the of intracellular calcium concentration were detected. Collectively, these results suggested that the phagocytic activity of PMO cells might be triggered by ODN via G protein/PKC signaling pathway, but not associated with the IP3/PKA/calcium pathway.
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