Study On Organic And Cellular Organizations Of Immune System And Some Effector Cells Of Paralichthys Olivaceus

As a lower vertebrate, although fish doesn't possess an immune system as good as primates, it do have fully developed its own innate and acquired immune system. Expanding of industrialized marine fish farming is accompanied with more and more invading microorganisms which cause serious problems both ecologically and economically in fish farming industry including the flounder, Paralichthys olivaceus. However, the technical resource for the control of aquatic diseases is extremely limited and efficient and ecological techniques are currently not available. Understanding of the fish immune system is basically a prerequisite both for solving the problem and for developing optimal epizootic diseases prevention and cure procedures in fish. The study performed the following basic researches on the flounder's immune system, including histological research of the immune organs (kidney, spleen and mesentery), functional analysis of peritoneal macrophages, and gene expression analysis of macrophages as well as other immune-related cells isolated from the kidney, spleen and peripheral blood. The purpose of the study is to provide scientific guidelines for diseases control in fish farming of the flounder.Histological methods were utilized in examining the structure of the major immune organs of the flounder, i.e. kidney, spleen and mesentery. The results showed the head kidney had a similar structure with the spleen in that both of them were comprised of a network formed by connection of reticular Cells-sinusoids-blood vessels, and the network supported migrations and responses initiated by lymphocytes and non-lymphocytes within. In addition, melanomacrophage center (MMC) was found to be a characteristic structure in both the spleen and the head kidney. Besides, after infection, the two organs responses in a much similar way, which was characterized by sinusoids expansion and increasing of flowing cell numbers, proliferation of the MMCs and formation of enhanced tight connection between MMC and sinusoids/blood vessels, formation or proliferation of ellipsoids and distinct border appeared between white and red pulp.There also existed some distinguished differences between them. The head kidney is a hematogenic tissue while the spleen is not. It appeared in the head kidney MMCs played an more important role in antigen uptake and transportation, while MMCs in spleen mainly functioned as scavengers. MMCs differentiated in function were also indicated by the fact that the MMC proliferation was much slower in the spleen than in the head kidney.The results indicated that meso- and meta-nephro were filled with glomeruli and renal tubules, and the mesonephro had a characteristic structure of 'middle vessel' which function was still unknown. Mesentery had a well developed sinusoid-vessel system. The main sinusoid was extremely large in size and stetched out to peritoneal cavity. Under infection, lots of macrophages moved toward peritoneal cavity.Therefore, structures of the spleen and the kidney of the flounder were similar to that of most known fish so far. The head kidney lacked in glomeruli and renal tubules and functioned as a hematogenic organ and a site for initiating immune responses. Sinusoids that run through meso- and meta-nephros containing numerous lymphocytes, leucocytes, mononuclear/macrophages, in addition to the MMCs distributed all over the organ strongly supported that the kidney was active in initiating immune responses as whole. Mesentery might account for the main source of induced peritoneal macrophages.By dual florescence labeling of heat-inactivated bacteria, Vibrio anguillarum, with FITC and EtBr, the liquid paraffin induced peritoneal macrophages was shown to possess a strong phagocytic capability by FACS detection. The phagocytic rate was 32% at 30 min and reached plateau phase of 55% at 75 min under temperature 18-22℃. Both in situ florescence and optical microscopic observation indicated that the induced peritoneal macrophages did indeed show a distinct phagocytic process. Thus, the peritoneal macrophages had a strong phagocytic capability against the heat-inactivated bacteria Vibrio anguillarum in vitro.Lymphocytes were successfully isolated from peripheral blood and the major immune organs including spleen, head kidney, meso- and meta-nephros via combined Percoll continuous density gradient with lydroxypropylmethyl cellulose separation. A set of primers were used to screen normal and LPS-stimulated gene expression pattern of the cells isolated. The target genes include genes encoding PAMP receptors (TLR 3 and 9), members of Janus kinase /signal transducer and activator of transcription (JAK/STAT) signal transduction pathway (IκBα, MyD88 and STAT1), antigen processing and display proteins (PA28a, MHC Ia and MHC IIP) and secreted IgM. RT-PCR detection showed peritoneal macrophages expressed genes involved in antigen recognition and signal transduction of the two TLR receptors, MyD88 and STAT1 stably, while the expression was insensitive to LPS stimulus. mRNA of secreted IgM could be detected in the cells isolated from PBL, spleen and head kidney, with increasing of the quantities over the time of stimulation. The results showed that the macrophages possessed receptors for PAMP (Pathogen-associated molecular patterns), and they could correspondingly utilize JAK/STAT pathway in signal transduction. Plasma B cells were either fewer in quantity or lower in antibody producing capability in the head kidney, while as the PBL or the spleen were the main site for antibody production.