| Four methods were used to classify the haemocyte of scallop (Chlamys farreri). By light microscope observation with Giemsa stain, hemocytes were stained purple. Hyalinocytes were agranular and showed central nucleus surrounded by relatively small cytoplasm, their nucleus/cytoplasm (N/C) ratio was high (0.64?.14). Granulocytes were more abundant than hyalinocytes and had several granules in endoplasm, furthermore, the size of granulocyte was larger and the endoplasm of granulocyte was denser than hyalinocyte, but their N/C ratio was low (0.35?.10). Some of granulocytes had double nuclei. Observed at the ultrastructural level by electron microscope, hyalinocyte had no organelles and contained abundant euchromatin. Granulocyte had a few organelles and had pseudopod. The cytoplasm of granulocyte was electron-dense and had membrane-limited granules. The two nuclei (double-nucleus) in granulocytes were similiar. By the way of density gradient centrifugation, the two type hemocytes were seperated: hyalinocytes mainly in upper interface, granulocytes in lower interface. With the method of flow cytometry, hemocyte cells were classified into hyalinocyte and granulocyte by forward and side light-scatter parameters analysis according to cell size and granule.The activities of peroxidase (POD), phenoloxidase (PO) and alkaline phosphatase (ALP) in haemocytes were analyzed by immunocytochemical and biochemical methods. By using the substrate of DAB, POD was found cytochemically as fine, brown reaction products throughout the cytoplasm in both types of haemocytes. Positive rate of POD was 49.3%, hyalinocyte was 18.6%, and granulocytes was 30.7%, meanwhile, hyalinocyte positive rate (HPR) of POD was 39%, granulocyte positive rate (GPR) was 47%. PO was observed as a fine gray to black granular deposit within the cytoplasm of the haemocytes by using L-dopa as substrate. Counterstaining with Wrights stain demonstrated that the PR of PO was 37.5%, hyalinocyte was 13.4%, and granulocyte was 24.1%, however, HPR of PO was 32%, GPR was 40%. By using NBT/BCIP as substrate,ALP occurred cytochemically in haemocyte, producing a fine blue granular deposit within the cytoplasm. Counterstaining with Wrights stain illustrated that PR of ALP was about 40.1%, hyalinocyte was 16.3%, and granulocyte was 23.8%, while HPR of ALP was 50%, GPR was 61%.By using DAB method, POD activity in different haemocyte types were shown. According to the absorbance of HRP as a standard, POD activity was 2.4 ng/ml in hyalinocytes, 3.9 ng/ml in granulocytes. Using L-dopa as substrate, PO activity occurred in both types of haemocyte, and it was 9 units in hyalinocytes, 14 units in granulocytes calculated by the protein content. Activity of ALP was shown by using pNPP method, and it was 3.0 u/1 in hyalinocytes, 20.9 u/1 in granulocytes. All the results indicate that POD, PO and ALP were more abundant and more active in granulocytes than in hyalinocytes.Employing NBT method, phagocytosis occurred in both types of haemocyte, and positive rate of haemocyte was 53.7%, comprising 18.2% hyalinocyte and 35.3% granulocytes. Antibacterial activities of hyalinocyte was 0.196, and granulocyte was 0.385. Bacteriolysis of hyalinocyte was 0.122, and granulocyte was 0.262. Lyzozyme activities of hyalinocyte was 0.80 mg/L, and granulocyte was 2.78 mg/L. When two type haemocyte suspensions were mixed with bacteria respectively, oxygen' intermediates production of granulocyte was stronger than hyalinocyte. It increased by density of bacteria, and reach the highest values when bacteria was 108cell/ml. Determination of antibacterial activities showed that the phagocytosis, bacteriolysis and oxygen intermediates production of the granulocytes were higher than those of the hyalinocytes.The Balb\C mice were immunized by heamocytes of Chlamys farreri. Spleens from the immunized mice were dissected and cells were fused with a P3-X63-Ag8Ul myeloma cell line using polyethylene glycol as fusogen. Hybridomaculture medium were assayed by immunofluorescence assay techniques (IFAT).
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