新型免疫增强剂——CpG DNA对鱼类免疫活性的调节作用
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摘要
近年来发现，细菌基因组DNA和人工合成的含有未甲基化“CpG,”motif的特定寡核苷酸序列(统称为CpG DNA)能够直接或间接刺激人和小鼠等多种高等动物的B淋巴细胞、单核细胞、巨噬细胞、树突状细胞等抗原递呈细胞以及天然杀伤细胞的活化或增殖，是一种具有广泛作用的免疫激活剂。但CpG DNA对鱼类免疫功能的调节作用研究尚刚起步。
本实验的第一部分，首次以草鱼体外培养的头肾巨噬细胞为模型，探讨了多种人工合成的寡核苷酸序列和两种不同细菌的基因组DNA对巨噬细胞活化的作用。实验中采用了含不同CpG motif的序列：在小鼠和人中有良好刺激作用的最适序列ODN-1826(GACGTT)和ODN-2006(GTCGTT)；在大西洋鲑中有刺激活性的ODN-1670(AACGTT)；在ODN-1670基础上设计的包含两个AACGTT重复的序列D以及带有反向“CG”的序列R。两种细菌DNA分别来自大肠杆菌JM109和青春双歧杆菌基因组。在实验中，我们发现含有CpG的寡脱氧核糖核苷酸序列(ODNs)和细菌DNA都能引起巨噬细胞的活化，主要表现为细胞内的O_2~-、H_2O_2水平、酸性磷酸酶释放的增加和杀菌能力的提高，并且体现出一定的剂量效应，但是反向序列R却不能引起这些变化。说明CpG DNA在增强鱼类细胞介导的免疫反应方面有重要的作用。有趣的是，我们还发现具有刺激作用的四种含有不同CpG motif的ODNs和两种细菌DNA对巨噬细胞活化作用之间并没有显著差异，说明鱼体对识别不同CpG DNA并不敏感，CpG DNA中未甲基化的“CG”对鱼类细胞的免疫刺激起主要作用。这些发现也提示我们，CpG DNA可以作为我们进一步理解鱼类是如何抵抗病原细菌侵染的重要工具。
论文的第二部分，首次研究了大肠杆菌DNA对草鱼体内各种重要的免疫相关因子表达水平的影响。这些因子包括MHCⅡ，前B细胞增强因子(pre-B-cell enhancing factor, PBEF)，天然杀伤细胞增强因子(natural killer cell enhancement factor, NKEF)和IFN诱生蛋白Mx。从经过大肠杆菌DNA体内注射48小时的草鱼头肾中分离总RNA，经过RT-PCR扩增获得MHCⅡα-2，PBEF, NKEF和Mx cDNA，与未注射的正常草鱼头肾组织相比较，发现PBEF，NKEF和Mx在诱导组织中大量表达，扩增片段清晰，而正常组织几乎扩增不出相应的
浙江大学硕士学位论文
cDNA。利用半定量RT一PCR，我们分析了MHC lla一2在诱导前后的含量变化，发
现大肠杆菌DNA的诱导能引起它表达量的显著增加，说明细菌基因组DNA能有
效激活草鱼免疫系统中一些重要因子的表达。
因此，我们认为，与在高等动物中的作用一样，CPG DNA也是鱼类免疫系统
的一种新型有效的免疫调节剂，它能增强鱼体内细胞和体液介导的免疫应答，从
而增强机体的抗病能力。同时它也将在减少鱼类病害和增强鱼用疫苗的免疫效力
方面有着广泛的应用前景。
In mice and humans, B cells, antigen-presenting cells (APCs) including monocytes, macrophages and dendritic cells and natural killer (NK) cells can be stimulated directly or indirectly by the bacterial DNA and oligodeoxynucleotides (ODNs) containing the CpG dinucleotides (CpG DNA).
In Part I, we used head kidney macrophages of grass carp (Ctenopharyngodon idellus) as an in vitro model and investigated the effects of several CpG-ODNs, Escherichia coli DNA and Bifidobacterium adolescentis DNA on fish immunocytes. The CpG-ODNs included the optimal motifs: the ODN-1826 (GACGTT) and -2006 (GTCGTT) for the mice and humans cells, the ODN-1670 (AACGTT) used in Atlantic salmon, the ODN-D containing two repeats motif of those in 1670 and the ODN-R with an inverted CpG. The results showed that CpG DNA has an immunomodulatory role on a dose-dependent way in grass carp, and all the ODNs and bacteria DNAs except the ODN-R could activate macrophages, increasing the levels of superoxide anion (O~2), hydrogen peroxide (H2O2), acid phosphatase and bactericidal activity. New evidence was provided that CpG DNA could induce the cell-mediated immunity in fish. Interestingly, no significant differences among the ODNs tested could be found and the ODN-D was not more efficient than 1670. There is also no remarkable differences between bacteria DNAs. It suggests that the sequence which contains the unmethylated 'CG' dinucleotides could make contribute to this immunostimulatory effect. These findings indicate that CpG DNA could be useful tools for understanding the important anti-bacterial defense mechanism in fish.
In Part II, we studied the effects of E.coli DNA on expression of some important immune-related cytokines including MHCⅡ, pre-B-cell enhancing factor(PBEF), natural killer cell enhancement factor(NKEF) and MX on grass carp. After 48h of E.coli DNA infection, we isolated total RNA of head kidney and used RT-PCR to amplified cDNA of MHCⅡ alpha-2,PBEF, NKEF and MX. Using semi-quartitative RT-PCR, we found the upregulated expression of MHC Ⅱ alpha-2 in CpG DNA-induced head kidneys compared with those of controls. It was shown that the remarkable bands of PBEF,
NKEF and MX from CpG DNA-stimulated fish, while there is no band tested from controls. It suggests that bacteria DNA may be an effective activator on immune moleculars of grass carp.
In a word, we observed that CpG DNA is a novel immunostimulatory in vivo and in vitro on fish as well as those on higher vertebrates, which promote the cell- and humoral-mediated immune responses to defense against the invading microorganisms. And it may have potential application in the minimizing the impact of fish diseases and enhancing the efficacy of antigen and DNA vaccines.
本实验的第一部分，首次以草鱼体外培养的头肾巨噬细胞为模型，探讨了多种人工合成的寡核苷酸序列和两种不同细菌的基因组DNA对巨噬细胞活化的作用。实验中采用了含不同CpG motif的序列：在小鼠和人中有良好刺激作用的最适序列ODN-1826(GACGTT)和ODN-2006(GTCGTT)；在大西洋鲑中有刺激活性的ODN-1670(AACGTT)；在ODN-1670基础上设计的包含两个AACGTT重复的序列D以及带有反向“CG”的序列R。两种细菌DNA分别来自大肠杆菌JM109和青春双歧杆菌基因组。在实验中，我们发现含有CpG的寡脱氧核糖核苷酸序列(ODNs)和细菌DNA都能引起巨噬细胞的活化，主要表现为细胞内的O_2~-、H_2O_2水平、酸性磷酸酶释放的增加和杀菌能力的提高，并且体现出一定的剂量效应，但是反向序列R却不能引起这些变化。说明CpG DNA在增强鱼类细胞介导的免疫反应方面有重要的作用。有趣的是，我们还发现具有刺激作用的四种含有不同CpG motif的ODNs和两种细菌DNA对巨噬细胞活化作用之间并没有显著差异，说明鱼体对识别不同CpG DNA并不敏感，CpG DNA中未甲基化的“CG”对鱼类细胞的免疫刺激起主要作用。这些发现也提示我们，CpG DNA可以作为我们进一步理解鱼类是如何抵抗病原细菌侵染的重要工具。
论文的第二部分，首次研究了大肠杆菌DNA对草鱼体内各种重要的免疫相关因子表达水平的影响。这些因子包括MHCⅡ，前B细胞增强因子(pre-B-cell enhancing factor, PBEF)，天然杀伤细胞增强因子(natural killer cell enhancement factor, NKEF)和IFN诱生蛋白Mx。从经过大肠杆菌DNA体内注射48小时的草鱼头肾中分离总RNA，经过RT-PCR扩增获得MHCⅡα-2，PBEF, NKEF和Mx cDNA，与未注射的正常草鱼头肾组织相比较，发现PBEF，NKEF和Mx在诱导组织中大量表达，扩增片段清晰，而正常组织几乎扩增不出相应的
浙江大学硕士学位论文
cDNA。利用半定量RT一PCR，我们分析了MHC lla一2在诱导前后的含量变化，发
现大肠杆菌DNA的诱导能引起它表达量的显著增加，说明细菌基因组DNA能有
效激活草鱼免疫系统中一些重要因子的表达。
因此，我们认为，与在高等动物中的作用一样，CPG DNA也是鱼类免疫系统
的一种新型有效的免疫调节剂，它能增强鱼体内细胞和体液介导的免疫应答，从
而增强机体的抗病能力。同时它也将在减少鱼类病害和增强鱼用疫苗的免疫效力
方面有着广泛的应用前景。
In mice and humans, B cells, antigen-presenting cells (APCs) including monocytes, macrophages and dendritic cells and natural killer (NK) cells can be stimulated directly or indirectly by the bacterial DNA and oligodeoxynucleotides (ODNs) containing the CpG dinucleotides (CpG DNA).
In Part I, we used head kidney macrophages of grass carp (Ctenopharyngodon idellus) as an in vitro model and investigated the effects of several CpG-ODNs, Escherichia coli DNA and Bifidobacterium adolescentis DNA on fish immunocytes. The CpG-ODNs included the optimal motifs: the ODN-1826 (GACGTT) and -2006 (GTCGTT) for the mice and humans cells, the ODN-1670 (AACGTT) used in Atlantic salmon, the ODN-D containing two repeats motif of those in 1670 and the ODN-R with an inverted CpG. The results showed that CpG DNA has an immunomodulatory role on a dose-dependent way in grass carp, and all the ODNs and bacteria DNAs except the ODN-R could activate macrophages, increasing the levels of superoxide anion (O~2), hydrogen peroxide (H2O2), acid phosphatase and bactericidal activity. New evidence was provided that CpG DNA could induce the cell-mediated immunity in fish. Interestingly, no significant differences among the ODNs tested could be found and the ODN-D was not more efficient than 1670. There is also no remarkable differences between bacteria DNAs. It suggests that the sequence which contains the unmethylated 'CG' dinucleotides could make contribute to this immunostimulatory effect. These findings indicate that CpG DNA could be useful tools for understanding the important anti-bacterial defense mechanism in fish.
In Part II, we studied the effects of E.coli DNA on expression of some important immune-related cytokines including MHCⅡ, pre-B-cell enhancing factor(PBEF), natural killer cell enhancement factor(NKEF) and MX on grass carp. After 48h of E.coli DNA infection, we isolated total RNA of head kidney and used RT-PCR to amplified cDNA of MHCⅡ alpha-2,PBEF, NKEF and MX. Using semi-quartitative RT-PCR, we found the upregulated expression of MHC Ⅱ alpha-2 in CpG DNA-induced head kidneys compared with those of controls. It was shown that the remarkable bands of PBEF,
NKEF and MX from CpG DNA-stimulated fish, while there is no band tested from controls. It suggests that bacteria DNA may be an effective activator on immune moleculars of grass carp.
In a word, we observed that CpG DNA is a novel immunostimulatory in vivo and in vitro on fish as well as those on higher vertebrates, which promote the cell- and humoral-mediated immune responses to defense against the invading microorganisms. And it may have potential application in the minimizing the impact of fish diseases and enhancing the efficacy of antigen and DNA vaccines.
引文
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LiuH一M，Newbrough SE，Bhatia SK，et al.Immunostimulatory CPG oligndenoxynueleotides enhanees the
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22
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eell resPonse.J Immunol 2000，165:4537一543.
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2000，247:211一225.