碩士 / 東吳大學 / 微生物學系 / 91 / Prophenoloxidase activating system ( PAS ) plays an important role in non-self recognition and host defenses, and is believed to be a defense indicator in crustaceans. In this study, haemocytes of giant freshwater prawn (Macrobrachium resenbergii) were in vitro treated with (1) CpG oligodexynucleotides (ODNs), LPS and laminarin for 30 min to determine the influence of the proPO system; (2) ODN 2006 to examine the ODN-bound cells and distribution of ODN binding protein of haemocytes; and (3) specific activators and/or inhibitors of certain signal transduction pathway to investigate the signaling of PAS.
In the experiment, raised extracellular POT detected in medium was regard as cell degranulation. Increase in both intracellular and extracellular POT was regard as new proPO synthesis. Following increased the concentration of LPS (60-80 μg/ml) and laminarin, the extracellular POT only slightly increased. PO activity measured in HLS treated with six of seven ODNs with different DNA sequence was significantly stronger than that by LPS or laminarin, except so-ODN 13. Following haemocytes treated with ODN 2006, both the intracellular and extracellular POS (P<0.05) were increased; however, only extracellular POT increased.
By observation using a fluoresent microscope, it was found that stimulated haemocytes without pseudopodia stretching were stained by fluorescent-label ODN; unstimulated cells were the same with stimulated cells. Using a confocal microscope, it was also observed that fluorescent intensity on the granules of unstimulated cells was higher than ODN-stimulated cells, but that on the cell membrane was lower than ODN-stimulated cells. It was speculated that haemocytes may express more ODN-binding protein on the surface membrane after ODN stimulation. Furthermore, by analysis of a flow cytometry, there was no difference in cell size and granule ratio between stimulated cells and unstimulated cells,.
In dot analysis, protein extract from haemocyte membrane or cytosol were reactive with ODN 2006. The result showed that the color signal of membrane fraction from either stimulated or unstimulated cells was higher. Therefore, it is suggested that membrane extract may have more relative amount of ODN-binding protein. Following analysis of protein electrophoresis and blotting, a 100 kDa ODN-binding protein was found.
In addition, the signal transduction pathways associated with the activation of PAS were determined. The results showed that the extracellular POT increased after extracellular medium separately treated with NaF (a G protein activator), caffeine (a phosphodiesterase inhibitor) and Phorbol-12- myristate-13-acetate (PMA) (a PKC activator), but decreased after treatment with either 8-bromo-cAMP (a PKA activator) or chelerythrine (a PKC inhibitor). Furthermore, extracellular POT resulted from extracellular medium co-treated with OND2006 and chelerythrine was lower than that stimulated with ODN, but was enhanced after treatment with genistein (an inhibitor of protein tyrosine kinase). Collectively, these results suggest that ODN receptor may be existed on surface and in cytosolic vesicles of prawn haemocytes, more receptors can be stimulated to present on cell surface by ODN stimulation, and the activation of proPO system, especial for degranulation, is triggered via protein kinase C and regulated via a signal pathway associated with tyrosine kinase.
| Identifer | oai:union.ndltd.org:TW/091SCU00381013 |
| Date | January 2003 |
| Creators | Che-Pei Chuo, 卓哲霈 |
| Contributors | Hung-Hung Sung, 宋宏紅 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 95 |
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