Influence On Immune Function Of Oligochitosan Stimulated Head Kidney Macrophages In Blunt Snout Bream(megalobrama Amblycephala)

Oligochitosan, which are the oligomer homologs of chitosan, are soluble and can be satisfactorily utilized, it has also been proved to possess versatile functional properties such as antitumor, antimicrobial activities and enhance the immunity. oligochitosan are able to enhance the function of immune system which is stimulate immune cell, such as macrophage、T lymphocytes, activated macrophages can function as immunoregulation by cytokines secretion,reactive oxygen and NO production. A number of studies have shown that oral administration of oligochitosan were all able to stimulate the innate immune responses of fish and could confer protection against infections, However the specific mechanism is unclear. Therefore we investigated the mechanisms underlying the immunostimulating effects of oligochitosan on the blunt snout bream in vitro and in vivo. Main results for this study are as follows:1 Blunt snout bream head-kidney macrophages were isolated、purified、characterized, Our statistic data showed that the purity of the macrophages from blunt snout bream was above 98%.2 The phagocytic activity of macrophages were measured by Elisa and Fluoresence microscope, we used E. coli-GFP for the assay, the results showed that macrophages stimulated with oligochitosan contained more bacteria in the cytoplasm of the macrophages than those in the control group. It was able to enhanced the superoxide anion、NO production、the activity of i NOS, m RNAs of i NOS and all five subunits(p91phox 、p22phox、p40phox、p47phox、p67phox)were significantly elevated.3 Blunt snout breams have been fed with feed supplemented with 1% oligochitosan for one week, macrophages derived from oligochitosan treated-fish showed significant higher respiratory burst activity and NO production ability. the m RNA levels of the TNF-a and IL-1β were also significantly increased.4 The full-length gci NOS contained 4215 bp, consisted of a 5’ terminal untranslated acid sequences showed that gci NOS uncontained a signal peptide, which contain four domain, NO synth domian(at positions 80 bp-451 bp); flavodoxin 1 domain(490 bp-621 bp); flavin adenine dinucleotide(FAD) binding domain(670 bp-897 bp); nicotinamide adenine dinucleotide(NAD) binding domain(712 bp-941 bp). 3’ end has the typical plus tail signal AATAAA and Poly A tail. TMpred software analysis showed that the gci NOS has three transmembrane domains composed of 28, 25 and 21 amino acid respectively, it also contains the cofactor-blinding sites for heme 、 calmodulin 、 tetrahydrobiopterin 、FADpyrophosphate、FMN、FADisoalbxazine、NADH.The results show that oligosaccharide could strengthen nonspecific immune response by improving the phagocytosis of macrophage. Activated macrophages could product reactive oxygen, reactive nitrogen and secrete cytokines(IL-1β、TNF-α), then activating the body’s nonspecific immune.