凡纳滨对虾血细胞免疫抑制剂的筛选与免疫调控作用的研究
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摘要
本文研究了凡纳滨对虾血细胞免疫抑制剂的筛选以及脂多糖与凝集素抑制剂联合作用对对虾的免疫调控作用。主要内容包括:(1)凡纳滨对虾(Litopenaeus vannamei)血细胞免疫活性培养方法的研究;(2)凡纳滨对虾血细胞酚氧化酶、凝集素抑制剂筛选方法的研究;(3)脂多糖与凝集素抑制剂联合作用对凡纳滨对虾酚氧化酶原级联系统的影响。主要研究结果如下:
1.凡纳滨对虾血细胞免疫活性培养方法的研究
本文通过小鼠、兔、鲈鱼和人的ABO七种红细胞与凡纳滨对虾血清凝集素发生凝集反应,选择凝集效价最高的小鼠红细胞测定对虾血细胞凝集活性;同时本文选择了四种培养基成分进行凡纳滨对虾血细胞离体培养,结果表明:四种培养基对凡纳滨对虾血细胞数量、存活率和酚氧化酶原级联系统胞吐影响显著(P<0.05),随着培养时间的延长,四种培养基各处理组血细胞数量、存活率和酚氧化酶原活力逐渐下降,而酚氧化酶活力逐渐升高。在12h内二号培养基总血细胞数量和血细胞存活率均显著高于一号、三号和四号培养基,四种培养基在3-6h内,血细胞存活率趋于稳定,6h之后逐渐下降;在0-3h内,培养基一号、三号和四号凝集活性升高,之后逐渐下降,而二号培养基3h内凝集活性没有发生变化,6h降低后趋于稳定;在0-6h内培养基一号酚氧化酶原活力高于其他各组,培养基二号酚氧化酶活力最低,在12h时培养基二号酚氧化酶原活力最高,酚氧化酶活力与其他三组无明显差异。由此得出结论,选择二号培养基进行凡纳滨对虾血细胞体外培养研究。
2.凡纳滨对虾血细胞酚氧化酶、凝集素抑制剂筛选方法的研究
结果表明:三种浓度的酚氧化酶抑制剂p-APMSF对凡纳滨对虾血细胞数量和存活率影响显著(P<0.05),随着培养时间的延长,各处理组血细胞数量、存活率逐渐下降。在6h内25μmol/L p-APMSF处理组显著高于50μmol/L和100μmol/L处理组;在1-6h内,25μm处理组血细胞数量变化不显著,但存活率差异显著;在实验时间内,各处理组凝集活性比对照组升高,处理组之间,50μm和100μm处理组凝集活性相同,25μm处理组凝集活性最低;25μm处理组酚氧化酶原活力和丝氨酸活力显著高于其他处理组,与p-APMSF浓度呈明显的负相关性。三种凝集素抑制剂对凡纳滨对虾血细胞数量和存活率影响显著(P<0.05),在实验时间内,各处理组对虾血细胞数量和存活率逐渐下降,在3-6h内,200mM葡萄糖处理组血细胞数量和存活率高于其他处理组,且变化不显著;各处理组酚氧化酶活力逐渐升高,而血细胞酚氧化酶原活力逐渐下降;葡萄糖处理组对凡纳滨对虾凝集活性完全抑制作用。因此,选择葡萄糖作为凡纳滨对虾凝集素抑制剂进行相关研究。
3.脂多糖与凝集素抑制剂联合作用对凡纳滨对虾酚氧化酶原系统的影响
结果表明:脂多糖与凝集素抑制剂联合作用对凡纳滨对虾总血细胞数量(THC)酚氧化酶原proPO级联系统影响显著(P<0.05)。在实验时间内,各处理组血细胞数量和存活率均逐渐下降,与脂多糖处理组相比,脂多糖与凝集素抑制剂联合作用处理组血细胞数量和存活率低;与对照组相比,各处理组凡纳滨对虾凝集活性降低,3h内LPS处理组与对照组对虾凝集活性升高,而脂多糖与凝集素抑制剂联合作用处理组凝集活性降低;3-6h内,各处理组对虾凝集活性无显著变化;脂多糖、脂多糖与凝集素抑制剂联合作用对凡纳滨对虾胞吐酚氧化酶活力以及血细胞酚氧化酶原活力、血细胞丝氨酸蛋白酶原活力影响显著(P<0.05)。在实验时间内,各处理组酚氧化酶原活力和丝氨酸蛋白酶原活力均下降,与对照组相比,脂多糖处理组下降的幅度大;而酚氧化酶活力均升高。且各处理组血细胞胞吐酚氧化酶活力与培养时间呈明显的正相关性,而血细胞丝氨酸蛋白酶原活力和血细胞酚氧化酶原活力则与培养时间呈明显的负相关性。由此说明,凝集素在凡纳滨对虾免疫防御中发挥重要作用。
This paper reports the immunosuppressant screening and immune regulation by lectin inhibitor in the White Shrimp Litopenaeus vannamei, discussed mainly the following theses: (1) Establishment of haemocyte immune activity cell culture in vitro of Litopenaeus vannamei; (2) phenoloxidase inhibitor and lectin inhibitor screening of haemocyte from white shrimp Litopenaeus vannamei; (3) The effect of combination of LPS and lectin inhibitors on prophenoloxidase activating system of haemocytes in Litopenaeus vannamei.
1. Establishment of haemocyte immune activity keeping methods in vitro of Litopenaeus vannamei
The effects of four media on haemocyte counts, viability and exocytosis of prophenoloxidase cascade system were examined. It was founded that all four media have notable effects on haemocyte counts, viability, and exocytosis of prophenoloxidase (proPO) cascade system (P<0.05). During 0-6 h, the haemocyte counts, viability and prophenoloxidase activity in four treatments declined gradually, while phenoloxidase activity climbed increasingly. However, the haemocyte counts and viability in second medium were significantly higher than those three media, during 3-6h, haemocyte counts of each medium were stabilized, then decreased gradually after 6h; in 0-3h, the hemagglutinating activity of the first、third and forth medium increased, the second one did not change, then kept stability after decreased at 6h, during 0-6h, prophenoloxidase activity of the first medium was higher than another ones, while the phenoloxidase activity was the lowest, at 12h prophenoloxidase activity of the second medium was highest, and there was no significant difference between the second and the other three groups. So, it’s suggested that the second medium can be chosen as a cell culture solution of Litopenaeus vannamei haemocyte.
2. The research of phenoloxidase and lectin inhibitor screening of Litopenaeus vannamei haemocyte
Effect of phenoloxidase and lectin inhibitor on haemocyte counts, viability and prophenoloxidase cascade system were studied. The three concentrations of phenoloxidase inhibitors p-APMSF had a significant influence on both haemocyte counts and viability of the Litopenaeus Vannamei. As the incubation time went on, the counts and viability of each management group decreased gradually. The hemagglutinating activity of the 50μm and 100μm groups remained the same, while the 25μm group was the least among the treated groups. The prophenoloxidase activity and serine proteinase activity of the 25μm group were much higher than the others and had significant negative correlation with p-APMSF'concentration. The three kinds of lectin inhibitors had a significant influence on haemocyte counts and viability of Litopenaeus Vannamei(P<0.05). The counts and viability in each treated group decrease gradually in the experimental time. Each treated group's phenoloxidase activity increased gradually while the prophenoloxidase activity of haemocyte decreased gradually. The glucose treated group was completely inhibited to the hemagglutinating activity of Litopenaeus Vannamei. Therefore, we choose glucose to do experiment to the shrimp lectin inhibitors.
3. Effect of combination of LPS and lectin inhibitor on the Prophenoloxidase cascade system of Litopenaeus Vannamei
The combination of LPS and lectin inhibitor had a great influence on prophenoloxidase cascade system of Litopenaeus Vannamei(P<0.05). In the experiment period, haemocyte counts and viability of each management group decreased gradually. Compared with the LPS treated group, the counts and viability of the combination of LPS and lectin inhibitor was lower. Compared with the control group, each treated group's hemagglutinating activity decreased. In 3 hours, the hemagglutinating activity of LPS treated group and the control group increased, while the hemagglutinating activity of the combination of LPS and lectin inhibitor decreased. During 3 to 6 h, the hemagglutinating activity of each group did not change much. LPS and combination of LPS and lectin inhibitor had a great influence on phenoloxidase activity, prophenoloxidase activity and serine protease activity of haemocytes (p<0.05). In the experiment period, both the proPO activity and Serine protease activity of each treated group decreased, and compared with the control group, LPS treated group decreased lower while all of the PO activity increased. In each treated group, phenoloxidase activity shows a positive correlation with the incubation time, while the serine protease activity and proPO activity were negatively correlated with the incubation time. Therefore, lectin plays an important role in the immune of Litopenaeus Vannamei.
1.凡纳滨对虾血细胞免疫活性培养方法的研究
本文通过小鼠、兔、鲈鱼和人的ABO七种红细胞与凡纳滨对虾血清凝集素发生凝集反应,选择凝集效价最高的小鼠红细胞测定对虾血细胞凝集活性;同时本文选择了四种培养基成分进行凡纳滨对虾血细胞离体培养,结果表明:四种培养基对凡纳滨对虾血细胞数量、存活率和酚氧化酶原级联系统胞吐影响显著(P<0.05),随着培养时间的延长,四种培养基各处理组血细胞数量、存活率和酚氧化酶原活力逐渐下降,而酚氧化酶活力逐渐升高。在12h内二号培养基总血细胞数量和血细胞存活率均显著高于一号、三号和四号培养基,四种培养基在3-6h内,血细胞存活率趋于稳定,6h之后逐渐下降;在0-3h内,培养基一号、三号和四号凝集活性升高,之后逐渐下降,而二号培养基3h内凝集活性没有发生变化,6h降低后趋于稳定;在0-6h内培养基一号酚氧化酶原活力高于其他各组,培养基二号酚氧化酶活力最低,在12h时培养基二号酚氧化酶原活力最高,酚氧化酶活力与其他三组无明显差异。由此得出结论,选择二号培养基进行凡纳滨对虾血细胞体外培养研究。
2.凡纳滨对虾血细胞酚氧化酶、凝集素抑制剂筛选方法的研究
结果表明:三种浓度的酚氧化酶抑制剂p-APMSF对凡纳滨对虾血细胞数量和存活率影响显著(P<0.05),随着培养时间的延长,各处理组血细胞数量、存活率逐渐下降。在6h内25μmol/L p-APMSF处理组显著高于50μmol/L和100μmol/L处理组;在1-6h内,25μm处理组血细胞数量变化不显著,但存活率差异显著;在实验时间内,各处理组凝集活性比对照组升高,处理组之间,50μm和100μm处理组凝集活性相同,25μm处理组凝集活性最低;25μm处理组酚氧化酶原活力和丝氨酸活力显著高于其他处理组,与p-APMSF浓度呈明显的负相关性。三种凝集素抑制剂对凡纳滨对虾血细胞数量和存活率影响显著(P<0.05),在实验时间内,各处理组对虾血细胞数量和存活率逐渐下降,在3-6h内,200mM葡萄糖处理组血细胞数量和存活率高于其他处理组,且变化不显著;各处理组酚氧化酶活力逐渐升高,而血细胞酚氧化酶原活力逐渐下降;葡萄糖处理组对凡纳滨对虾凝集活性完全抑制作用。因此,选择葡萄糖作为凡纳滨对虾凝集素抑制剂进行相关研究。
3.脂多糖与凝集素抑制剂联合作用对凡纳滨对虾酚氧化酶原系统的影响
结果表明:脂多糖与凝集素抑制剂联合作用对凡纳滨对虾总血细胞数量(THC)酚氧化酶原proPO级联系统影响显著(P<0.05)。在实验时间内,各处理组血细胞数量和存活率均逐渐下降,与脂多糖处理组相比,脂多糖与凝集素抑制剂联合作用处理组血细胞数量和存活率低;与对照组相比,各处理组凡纳滨对虾凝集活性降低,3h内LPS处理组与对照组对虾凝集活性升高,而脂多糖与凝集素抑制剂联合作用处理组凝集活性降低;3-6h内,各处理组对虾凝集活性无显著变化;脂多糖、脂多糖与凝集素抑制剂联合作用对凡纳滨对虾胞吐酚氧化酶活力以及血细胞酚氧化酶原活力、血细胞丝氨酸蛋白酶原活力影响显著(P<0.05)。在实验时间内,各处理组酚氧化酶原活力和丝氨酸蛋白酶原活力均下降,与对照组相比,脂多糖处理组下降的幅度大;而酚氧化酶活力均升高。且各处理组血细胞胞吐酚氧化酶活力与培养时间呈明显的正相关性,而血细胞丝氨酸蛋白酶原活力和血细胞酚氧化酶原活力则与培养时间呈明显的负相关性。由此说明,凝集素在凡纳滨对虾免疫防御中发挥重要作用。
This paper reports the immunosuppressant screening and immune regulation by lectin inhibitor in the White Shrimp Litopenaeus vannamei, discussed mainly the following theses: (1) Establishment of haemocyte immune activity cell culture in vitro of Litopenaeus vannamei; (2) phenoloxidase inhibitor and lectin inhibitor screening of haemocyte from white shrimp Litopenaeus vannamei; (3) The effect of combination of LPS and lectin inhibitors on prophenoloxidase activating system of haemocytes in Litopenaeus vannamei.
1. Establishment of haemocyte immune activity keeping methods in vitro of Litopenaeus vannamei
The effects of four media on haemocyte counts, viability and exocytosis of prophenoloxidase cascade system were examined. It was founded that all four media have notable effects on haemocyte counts, viability, and exocytosis of prophenoloxidase (proPO) cascade system (P<0.05). During 0-6 h, the haemocyte counts, viability and prophenoloxidase activity in four treatments declined gradually, while phenoloxidase activity climbed increasingly. However, the haemocyte counts and viability in second medium were significantly higher than those three media, during 3-6h, haemocyte counts of each medium were stabilized, then decreased gradually after 6h; in 0-3h, the hemagglutinating activity of the first、third and forth medium increased, the second one did not change, then kept stability after decreased at 6h, during 0-6h, prophenoloxidase activity of the first medium was higher than another ones, while the phenoloxidase activity was the lowest, at 12h prophenoloxidase activity of the second medium was highest, and there was no significant difference between the second and the other three groups. So, it’s suggested that the second medium can be chosen as a cell culture solution of Litopenaeus vannamei haemocyte.
2. The research of phenoloxidase and lectin inhibitor screening of Litopenaeus vannamei haemocyte
Effect of phenoloxidase and lectin inhibitor on haemocyte counts, viability and prophenoloxidase cascade system were studied. The three concentrations of phenoloxidase inhibitors p-APMSF had a significant influence on both haemocyte counts and viability of the Litopenaeus Vannamei. As the incubation time went on, the counts and viability of each management group decreased gradually. The hemagglutinating activity of the 50μm and 100μm groups remained the same, while the 25μm group was the least among the treated groups. The prophenoloxidase activity and serine proteinase activity of the 25μm group were much higher than the others and had significant negative correlation with p-APMSF'concentration. The three kinds of lectin inhibitors had a significant influence on haemocyte counts and viability of Litopenaeus Vannamei(P<0.05). The counts and viability in each treated group decrease gradually in the experimental time. Each treated group's phenoloxidase activity increased gradually while the prophenoloxidase activity of haemocyte decreased gradually. The glucose treated group was completely inhibited to the hemagglutinating activity of Litopenaeus Vannamei. Therefore, we choose glucose to do experiment to the shrimp lectin inhibitors.
3. Effect of combination of LPS and lectin inhibitor on the Prophenoloxidase cascade system of Litopenaeus Vannamei
The combination of LPS and lectin inhibitor had a great influence on prophenoloxidase cascade system of Litopenaeus Vannamei(P<0.05). In the experiment period, haemocyte counts and viability of each management group decreased gradually. Compared with the LPS treated group, the counts and viability of the combination of LPS and lectin inhibitor was lower. Compared with the control group, each treated group's hemagglutinating activity decreased. In 3 hours, the hemagglutinating activity of LPS treated group and the control group increased, while the hemagglutinating activity of the combination of LPS and lectin inhibitor decreased. During 3 to 6 h, the hemagglutinating activity of each group did not change much. LPS and combination of LPS and lectin inhibitor had a great influence on phenoloxidase activity, prophenoloxidase activity and serine protease activity of haemocytes (p<0.05). In the experiment period, both the proPO activity and Serine protease activity of each treated group decreased, and compared with the control group, LPS treated group decreased lower while all of the PO activity increased. In each treated group, phenoloxidase activity shows a positive correlation with the incubation time, while the serine protease activity and proPO activity were negatively correlated with the incubation time. Therefore, lectin plays an important role in the immune of Litopenaeus Vannamei.
引文
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