栉孔扇贝(Chlamys farreri)血淋巴中一氧化氮和一氧化氮合酶的研究

STUDY ON NITRIC OXIDE AND NITRIC OXIDE SYNTHASE IN HAEMOLYMPH OF SCALLOP CHLAMYS FARRERI

采用组织化学、免疫组化和生物化学方法对栉孔扇贝血淋巴中的一氧化氮和一氧化氮合酶(EC1.14.13.39;NOS)进行了研究。结果表明,大部分血细胞呈NOS组化阳性,少量呈强阳性或弱阳性。神经型一氧化氮合酶(nNOS)和内皮型一氧化氮合酶(eNOS)在大部分血细胞呈阳性,且多在其细胞质局部集中分布;诱生型一氧化氮合酶(iNOS)在大部分血细胞也呈阳性,但多较均匀分布。分光光度测定结果表明,血清和血细胞中均存在NOS和NO,结构型一氧化氮合酶(cNOS,nNOS+eNOS)活力分别为2.87U和2.61U;iNOS活力分别为0.32U和0.45U;NO的含量分别为33.64和41.67μmol/g蛋白。病毒感染后,血清和血细胞中总一氧化氮合酶(tNOS)活力实验组均高于对照组,3h时差异显著,9h和24h时差异极显著;血清和血细胞中iNOS活力在3h、9h和24h时,实验组均高于对照组,且均差异极显著;血清和血细胞中NO含量在3h、9h和24h时,实验组也均高于对照组,且均差异极显著。栉孔扇贝血淋巴中存在的NO NOS体系可在抵抗病原生物感染方面发挥重要的作用。

Nitric oxide (NO), which is produced from L-arginine by nitric oxide synthase (EC1.14.13.39; NOS), act as a regulating agent in many areas such as immune system, nervous, and cardiovascular. In this study, the NOS in the haemocytes of scallop Chlamys farreri (sampled from Zhifu Bay, on the north of Yellow Sea; shell length 45—50mm) was studied with histochemical and immunohistochemical localization method. Activity of NOS and content of NO in the serum and haemocytes of normal scallop and the spherical viruses treated scallop were analyzed in spectrophotometric assay. The virus were isolated from infected C. farreri in a severe mortality in Jiaozhou Bay, and kept in stock in the Key Laboratory for Sustainable Utilization of Marine Fisheries Resource, China's Ministry of Agriculture. The aim of this study is to determine the roles that NO and NOS play in immunological defensing and regulating. The histochemical analysis of haemocytes showed that most of the haemocytes presented positive reaction to NOS and a few of them presented strongly or weakly positive reaction to NOS. The immunohistochemical analysis of the haemocytes showed that both neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) were detected in most of haemocytes. The stained granules were clumped in the cytoplasm; inducible nitric oxide synthase (iNOS) were detected in most of haemocytes too; and the stained granules were uniform in the cytoplasm. The biochemical test showed that both NOS and NO were detected in the serum and haemocytes. Enzyme activities of constitutive nitric oxide synthase (cNOS, nNOS+eNOS) in the serum and haemocytes were 2.87 and 2.61U respectively. The enzyme activities of iNOS in the serum and haemocytes were 0.32 and 0.45U respectively. The contents of NO in the serum and haemocytes were 33.64 and 41.67μmol/gprot respectively. After treated with the spherical viruses, the biochemical test results were as follow. The levels of total nitric oxide synthase (tNOS) activity of experimental groups both in the serum and haemocytes were higher than the control groups, the differences between experimental groups and control groups were significant at 3h, extremely significant at 9h and 24h. Levels of iNOS activity of experimental groups in the serum and haemocytes were significantly higher than control groups' at 3h, 9h and 24h. The contents of NO of experiment groups in the serum and haemocytes were significantly higher than control groups' at 3h, 9h and 24h. In summary, there are NO-NOS system in the haemolymph of the scallop C. farreri. The content of NO and the activity of NOS in haemolymph have significant changes after the animal was treated with virus. Results suggest that the NO-NOS system in haemolymph of the scallop plays an important role in its immunological defense.