摘要
为了解不同细菌刺激后仿刺参(Apostichopus japonicus)体腔液中免疫因子的应答变化,分别用灿烂弧菌(Vibrio splendidus)、哈维氏弧菌(V.harveyi)、假交替单胞菌(Pseudoalteromonas nigrifacien)、溶壁微球菌(Micrococcus lysodeikticus)和停乳链球菌(Streptococcus dysgadysgalactiae)注射刺激仿刺参,然后分别采用对硝基苯基磷酸酯(p NPP)底物法、氯化硝基四氮唑蓝(NBT)法、溶壁微球菌粉法和多巴络合物生成法对体腔液上清中的酸性磷酸酶(ACP)与碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)、溶菌酶(LYZ)和酚氧化酶(PO)的活力进行了测定。结果显示,灿烂弧菌刺激后,酸性磷酸酶和碱性磷酸酶活力显著升高,而超氧化物歧化酶、溶菌酶和酚氧化酶活力显著降低;哈维氏弧菌刺激后,酸性磷酸酶、超氧化物歧化酶、溶菌酶和酚氧化酶活力显著升高,碱性磷酸酶活力变化不规律;假交替单胞菌刺激后,酸性磷酸酶、溶菌酶和酚氧化酶活力显著升高,超氧化物歧化酶活力先升高后降低,碱性磷酸酶活力变化不规律;溶壁微球菌刺激后,酸性磷酸酶和酚氧化酶活力显著升高,超氧化物歧化酶活力先升高后降低,溶菌酶活力先升高后降低,而后在72 h恢复至对照水平,碱性磷酸酶活力变化不规律;停乳链球菌刺激后,除碱性磷酸酶活力在4 h有所下降外,其余免疫相关酶活力均显著升高。研究结果表明,酚氧化酶是仿刺参非特异性免疫系统中最敏感、高效的免疫因子之一;革兰氏阳性细菌与革兰氏阴性细菌之间在诱导仿刺参免疫因子应答变化上无明显规律性差异;溶壁微球菌诱导溶菌酶的应答变化与灿烂弧菌、哈维氏弧菌、假交替单胞菌和停乳链球菌存在明显差异,溶菌酶可能是仿刺参清除入侵溶壁微球菌的主要免疫因子;灿烂弧菌诱导仿刺参免疫因子应答变化显著不同于其他4株细菌,显示出本研究选取的5个免疫指标在预警灿烂弧菌病害上具有潜在应用价值;停乳链球菌在仿刺参中具有作为免疫增强剂的潜在应用价值。
Our previous study showed that the supernatant of coelomic fluid from sea cucumber（Apostichopus japonicus） exhibited strong antibacterial activity to Micrococcus lysodeikticus, but had no obvious effects on the growth of Vibrio splendidus, V. harveyi, Pseudoalteromonas nigrifacien and Streptococcus dysgadysgalactiae. In order to study the variation of immune-related enzyme activities in A. japonicus coelomic fluid after different bacterial challenges, five bacteria（bacterial information was listed in Table 1） described above were injected into A. japonicus, respectively, and then the activities of acid phosphatase（ACP） and alkaline phosphatase（AKP）, superoxide dismutase（SOD）, lysozyme（LYZ） and phenoloxidase（PO） were determined using p-nitrophenyl phosphate（p NPP） method, nitro blue tetrazolium chloride（NBT） method, M. lysodeikticus powder method and dopachrome formation method, respectively. The experiments of enzymatic activities determination were performed in quadruplication, and the data were expressed as means ± standard deviations（SD）. Statistical analysis was carried out using the software SPSS 11.5. Differences in enzymatic activities between bacteria-challenged group and the control group were analyzed by one-way ANOVA. The level of significance was defined as P 0.05. As shown in Fig. 1﹣5, the challenge of V. splendidus induced significant increase of acid phosphatase and alkaline phosphatase activities, but great decrease of superoxide dismutase, lysozyme and phenoloxidase activities; challenge with V. harveyi caused significant increase of acid phosphatase, superoxide dismutase, lysozyme and phenoloxidase activities and irregular variation of alkaline phosphatase activity; after challenge with P. nigrifacien, acid phosphatase, lysozyme and phenoloxidase activities increased significantly, superoxide dismutase activity increased firstly, and then decreased, and alkaline phosphatase activity exhibited irregular variation; after challenge with M. lysodeikticus, acid phosphatase and phenoloxidase activities increased, superoxide dismutase and lysozyme activities increased firstly, and then decreased, however, lysozyme activity returned to control level at 72 h post challenge, and alkaline phosphatase activity also exhibited irregular variation; the challenge of S. dysgadysgalactiae resulted in an inhibition on alkaline phosphatase activity at 4 h, but notable promotion on the activities of other determined enzymes. The results suggested that phenoloxidase was one of the most sensitive and efficient immune factors in A. japonicus innate immune system; no regular differences in the induction of A. japonicus immune factors were observed between Gram-positive bacteria and Gram-negative bacteria; between M. lysodeikticus and other four determined bacteria, the most significant difference was observed in the induction of lysozyme, and lysozyme might be the principle immune factor involved in the extermination of M. lysodeikticus; the variation of immune factors induced by V. splendidus differed greatly from those induced by other four bacteria, and the five immune factors selected in this study had potentials in the application for early warning of vibrio diseases; S. dysgadysgalactiae had potential in the application for immunostimulants in A. japonicus.
出处
《动物学杂志》
CAS
CSCD
北大核心
2015年第6期947-956,共10页
Chinese Journal of Zoology
基金
国家自然科学基金项目(No.31402336)
辽宁省科技计划项目(No.2015103044)
辽宁省海洋与渔业厅项目(No.201301
201502)
关键词
仿刺参
体腔液
细菌
免疫相关酶
Sea cucumber（Apostichopus japonicus）
Coelomic fluid
Bacteria
Immune-related enzymes
