博士 / 國立臺灣海洋大學 / 食品科學系 / 94 / The aims of this research are to investigate the hydrolytic activity of cellulase on chitosan, and to evduate in vitro and in vivo the immunomodulation activity of the hydrolytic products. The optimal reaction pH and temperature for cellulase digestion of chitosan with 95% deacetylation degree (DD95) were pH 5.2 and 55oC, respectively. Its hydrolystic activity increased with the increasing DD of chitosan. Using fed-batch technique, the productivity of the chitosan hydrolysate was increased by 1.3 times, if half amount of raw material was re-loaded after digestion of DD95 chitosan with cellulase for 1 h. Based on the function on the proliferation of a human hybridoma cell HB4C5, the hydrolysate from 9 h digestion was shown to have the highest activity. This hydrolysate is composed of a low-molecular-weight chitosan (LMWC) with a molecular mass of 20.0 kDa, and the chitooligosaccharide mixture (oligomixture), which are composed of sugars with degrees of polymerization of 1 to 8. In vitro, the chitosan hydrolysate increased both cell proliferation and IgM secretion of HB4C5 due to the presence of oligomixture for former activity and LMWC for the latter activity. All of the chitosanolytic products enhanced the production of superoxide anion in THP-1 monocytes. By intraperitoneal injection into mice, samples of the chitosan hydrolysate, oligomixture and LMWC significantly increased the levels of serum IgG and IgM, and enhanced the concanavalin A- and lipopolysaccharide- induced proliferation of mouse lymphocytes. Furthermore, the antitumor acitivty of the chitosanolytic products were studied in this research. All of the chitosanolytic products did not inhibit the proliferation of the tumor cells of MCF-7, HeLa, and HT-29, whereas both chitosan hydrolysate and LMWC significantly inhibited the growth of human leukemia cell HL-60 and U-937. The production of superoxide anion in HL-60 cells treated with LMWC was increased by 2.8 times compared to control. The facts that LMWC induced HL-60 cells to arrest in G0/G1 phase, and to express CD11b and CD14 markers on cells demonstrated that LMWC could induce the differentiation of HL-60 leukemia cells. All samples of the hydrolysate, LMWC and the oligomixture did not affect the proliferation of RAW264.7; while the hydrolysate and oligomixture elicited the phagocytic activity of RAW264.7 by 3.0 and 2.8 times, respectively. After treatment with the hydrolysate and oligomixture, the RAW264.7 macrophages became dendritic-like morphologic maturation with decreasing the oxidative burst of RAW264.7 by 30% and 15%, respectively. The oligomixture, which is the functional component in the chitosan hydrolysate, in combination with IFN-γ synergistically induced NF-κB activation, iNOS expression and NO production via binding with the receptors of CD14, TLR4 and CR3 receptors, followed by activation of the signal transduction systems of PI3K and MAPK (JNK and ERK) in RAW264.7 macrophages.
All samples of the hydrolysate, LMWC and the oligomixture were demonstrated to modulate non-specific and OVA-specific immunity after they were administrated into BALB/c mice at the dose of 500 mg/kg for 4 weeks. The phagocytic activity of the peritoneal macrophage was significantly increased when the mouse was fed with hydrolysate and oligomixture. The chitosan hydrolytic products were found to induce predominantly Th1 response. They also significantly enhanced concanavalin A (ConA)-stimulated proliferation of splenocytes and Peyer’s patch lymphocytes, and increased serum antibodies.
| Identifer | oai:union.ndltd.org:TW/094NTOU5253032 |
| Date | January 2006 |
| Creators | Guan-James Wu, 吳冠政 |
| Contributors | Guo-Jane Tsai, 蔡國珍 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 225 |
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