碩士 / 東吳大學 / 微生物學系 / 94 / 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 (Hernandez-Lopez et al., 1996; Le Moullac, 1998; Rodriguez & Moullac, 2000). Chuo et al., (2004) found that PAS can be triggered via either G protein/protein kinase C (PKC) or cAMP pathway. In (the research of) mammalian, signal pathway of CpG ODN stimulation starting from the acidification of endosome (Rutz et al., 2004), then through the NF-κB pathway, causes a series of defense gene performances.
The first step of the study is to investigate the influence of CpG ODN on phagocytosis activity and its signal pathway in Macrobrachium rosenbergii hemocytes. Subsequently, ODN 2006 is found to be able to enhance the respiratory burst of hemocyte, but reduced by Caffeine, the phosphodiesterase inhibitor which inhibits the degradation of cAMP. Cells treated with NaF, the G protein activator, and protein kinase C (PKC) activator, the Phenol-12-myristate-13-acetate (PMA), both enhance respiratory burst activity. The PKC inhibitor, Chelerythrine, could inhibit the respiratory burst, and also reduce the effect of PMA and ODN in respiratory burst activity.
In order to understand the relationship of endosome acidifications in CpG ODN activate PAS and phagocytosis pathway; the phagocytosis inhibitor, Chloroquine, and anti-MyD88 antibody are employed. Chloroqune could reduce the extracelluler POT activated by ODN2006, but has no effect to respiratory burst. Cells were labeled with Fluorescent-labeled anti-MyD88 antibody, detected by flowcytometer. The result showed that most cells have fluorescent intensity. Using fluorescent microscope to observe the co-location of MyD88 and ODN, it only finds the signal of ODN. In western blot analysis, it is not able to find the MyD88 molecular either.
To further understand the signal pathway of CpG ODN activate PAS, using Pyrrolidine dithiocarbamate (PDTC), the inhibitor of NF-κB, could inhibit extarcellular POT. Cells treated with the ERK inhibitor, PD98059, 50 % of the blood cell sample are not inhibited, and another 50% has no obvious variety. Using NF-κB signal pathway related to molecular antibody to examine the difference of signal molecular inside the hemocyte whether stimulates or not by CpG ODN, and no signal were detected.
Besides, signal molecular difference of CpG ODN stimulation was detected, but NF-κB signal pathway related to molecular antibody was not detected any signal. For cloning IκB cDNA, several degenerate primers were used. PCR products were in sequence and compared with fruit fly Cactus nucleotide sequence, the result showed that the similarity of four fragments were 71.4 %、60.0 %、56.6 % and 68.7 %.
Comprehensive above-mentioned result, CpG ODN may enhance phogocytosis via G protein and PKC or cAMP pathway, and this signal pathway has nothing to do with endocytosis. However, phagocytosis may not only participate in the signal transduction of CpG ODN activates PAS activity, but also involve in signal pathway of the NF-κ B.
| Identifer | oai:union.ndltd.org:TW/094SCU05381008 |
| Date | January 2006 |
| Creators | Pang-hung Chen, 陳邦鴻 |
| 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 | 110 |
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