文章摘要
外源因子介导调控ROS对中国鲎血淋巴细胞参数的影响
Effect of ROS mediated regulation by exogenous factors on lymphocyte parameters of Tachypleus tridentatus
投稿时间:2020-11-06  修订日期:2020-12-19
DOI:
中文关键词: 中国鲎  N-乙酰半胱氨酸  灭活鳗弧菌  血淋巴细胞  血化学指标
英文关键词: Horseshoe crab  N-acetylcysteine  Vibrio anguillarum  Lymphocyte  Blood chemical index
基金项目:2017 北海市“ 十三五” 海洋经济创新发展示范项目“ 鲎试剂及中华鲎生态利用产业链协同创新项目”(Bhsfs006)资助
作者单位邮编
徐镇 上海海洋大学水产与生命学院 201306
江宁锦 上海海洋大学水产与生命学院 201306
何锦锋 北海市产品质量检验所 
台宗光 上海市皮肤病医院 
朱全刚 上海市皮肤病医院 
王有基 上海海洋大学水产与生命学院 
胡梦红 上海海洋大学水产与生命学院 201306
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中文摘要:
      以鲎血为主要原料制备鲎试剂是我国鲎资源开发的关键产业链。我国鲎种群数量逐年下降,为实现鲎资源的保护和可持续利用,开展对中国鲎造血作用机理的相关研究刻不容缓。本研究向中国鲎体内注射N-乙酰半胱氨酸(NAC)和灭活鳗弧菌(V),比较注射后0, 6, 12, 24, 48小时(h)中国鲎的血淋巴细胞总数、活性氧含量及非特异性免疫酶活性变化。结果表明,与对照组相比,注射NAC后血淋巴细胞总数(THC)、活性氧(ROS)、丙二醛(MDA)活性均有下降趋势,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、总抗氧化酶(T-AOC)、溶菌酶(LZM)、碱性磷酸酶(AKP)活性均有升高趋势。NAC与不同浓度V共同刺激下,THC、ROS、MDA含量相对仅注射NAC的下降有所减缓,其它酶活性有所升高。而血蓝蛋白(HC)在整个实验中无明显变化。6-48h,NAC组的THC、ROS与V组、NAC和V共刺激组相比呈降低趋势,共同刺激下SOD活性显著高于其它组。48h,NAC、V及共刺激下CAT、T-AOC组间无显著差异,但均高于对照组。NAC+106V的MDA含量在48h最低,AKP活性在12-48h呈升高趋势,而NAC组的LZM活性在48h最高。注射NAC可降低THC、ROS,共刺激可缓和下降,V组THC、ROS升高,但其余血淋巴参数均提高,由此推测NAC和V均能刺激中国鲎血淋巴细胞的免疫机能;机体内的ROS含量对中国鲎血淋巴细胞增殖及再生起着重要作用。
英文摘要:
      There are three genera and four species of horseshoe crabs in the world. There are two species of Chinese horseshoe crabs and round tail horseshoe crabs in China.The tachypleus amebocyte lysate (TLA) production using horseshoe crab blood as the main raw material is the key industry chain in the utilization of horseshoe crab resources in China. So the demand for horseshoe crabs is increasing, the population of horseshoe crab is decreasing year by year in China. In order to achieve the protection and sustainable utilization of horseshoe crab resources, it is urgent to study the hematopoietic mechanism of the horseshoe crab, and to make the rapid recovery of hemopoietic lymphocytes from the horseshoe crab. Therefore, strengthening the recovery of horseshoe crabs blood lymphocytes is expected to reduce the loss of horseshoe crabs wild resource. In this study, male horseshoe crab were selected as experimental subjects. Eighteen male horseshoe crab with complete appendages, no parasitic and male horseshoe crabs of similar weight were randomly selected (initial body weight were 1375.57±150.24g,Precursor for wide were 26.41±1.49 cm). Randomly selected male horseshoe crab were divided into six groups with three individuals in each group. Different reagents were injected into the membrane on the side of the sixth step of horseshoe crab and the trial dose was 1 ml. ①0.85% normal saline、 ②150mg/ml N-acetylcysteine (NAC)、 ③105 mL-1 Vibrio anguillarum (105V)、 ④ 106 mL-1 Vibrio anguillarum (106V)、 ⑤ 0.5ml 150mg/ml N-acetylcysteine+ 0.5ml 105mL-1 Vibrio anguillarum (NAC+105V)、 ⑥ 0.5ml 150mg/ml N-acetylcysteine+ 0.5ml 106 mL-1 Vibrio anguillarum (NAC+106V). The experimental reagent N-acetylcysteine was dissolved in 0.85% sterile saline and prepared into the corresponding concentration of working fluid. Each set of horseshoe crabs were raised in a separate moving fish pond (2 meters long, 1 meters wide and 0.9 meters high), and the three horseshoe crabs in the pond were divided into different number of rubber bands to distinguish each one. The experiment period was two days, and the environmental conditions during the experiment were consistent with those during the temporary feeding. To observe the changes of reactive oxygen species and non-specific immune enzyme activity in total lymphocytes of horseshoe crabs at 0, 6, 12, 24 and 48 hours after injection were observed. During the experiment period, the horseshoe crab in each group did not die at the end of the experiment (48 hours), and the weight of each component was 1374.07±150.26g, the precursor width is 26.39±1.50cm, there was no significant difference from the beginning of the experiment (0 hours) (P>0.05). The results showed that compared with the control group, the total hemocyte counts (THC), reactive oxygen species (ROS), malonyldialdehyde (MDA) of horseshoe crabs after NAC injection showed downward trends, while the activity of superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), lysozyme (LZM), and alkaline phosphatase (AKP) showed upward trends. Under the stimulation of N-acetylcysteine and Vibrio anguillarum at different concentrations, the decrease trend of THC、ROS、MDA in horseshoe crab was slowed down compared with single injection of NAC, while the activities of other enzymes were increased. However, HC showed no significant change over the course of the experiment. At 6-48 hours, THC, ROS in the N-acetylcysteine group showed a decreasing trend compared with the Vibrio anguillarum group and Co-stimulation groups between N-acetylcysteine and Vibrio anguillarum, the SOD activity of N-acetylcysteine and Vibrio anguillarum was significantly higher than other groups. At 48 hours, CAT, T-AOC was no significant difference at the N-acetylcysteine group, Vibrio anguillarum group and Co-stimulation groups, but both of them were significantly higher than the control group. The NAC+106V group had the lowest MDA in 48 hours. The activity of AKP increased from 12 to 48 hours, and the activity of LZM in N-acetylcysteine group was the highest at 48 hours. Moreover through experimental comparison and analysis, it is concluded that r=0.8015 and P<0.0001 in the scatter diagram between ROS content and THC show a very significant and strong linear positive correlation. The principal component analysis of hemolymph parameters of Horseshoe crab showed that during the experiment, the two principal components of different treatment groups and time accounted for 86.52% of the total components, which had a significant main effect on immunity. PC1 accounts for 64.75 percent of the variance, the main effect on immunity is significant, the effects of different treatment groups on the immune function parameters of hemolytic lymphocytes were described, indicating that there were significant differences between the control group and the different treatment groups. This represents the specific response under different treatment conditions of N-acetylcysteine and inactivated Vibrio anguillarum, as the control group is horizontally isolated from the other treatment groups. PC2 accounts for 21.77 percent of the variance, This represents the specific response at different times in different treatment groups, as the N-acetylcysteine and the inactivated Vibrio anguillarum treatment group and the control group are isolated on this axis. The results show that there is a negative correlation between THC、ROS、MDA、HC and SOD、CAT、T-AOC、LZM、AKP. N-acetylcysteine can lower the value of horseshoe crab THC、ROS、NAC and Co-stimulation with the Vibrio anguillarum can mitigate the decline, However, the above results showed thatinactivated Vibrio anguillarum can increase THC、ROS of horseshoe crab,and antioxidant and immunoenzyme activities were increased in all treatment groups. Therefore, it can be concluded that N-acetylcysteine can reduce THC、ROS of horseshoe crab and co-stimulation of N-acetylcysteine with Vibrio anguillarum can alleviate the decreasing trend,however, inactivated Vibrio anguillarum could increase THC、ROS of horseshoe crab, and the activity of antioxidant and immunoenzyme was increased in all treatment groups. This indicated that when N-acetylcysteine reduced ROS content in horseshoe crab, hemolytic cells in the body also decreased, while when N-acetylcysteine combined with Vibrio anguillarum inactivated, ROS、THC decreased less than the N-acetylcysteine group. It can be concluded that N-acetylcysteine and Vibrio anguillarum can stimulate the immune functions of horseshoe crabs, meanwhile, ROS may play an important role in the proliferation and regeneration of the horseshoe crabs.
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