|四种类型CpG ODNs 对水产养殖甲壳动物免疫调节作用的初步研究|
|其他题名||Preliminary study on the immunomodulatory mechanism of different CpG ODNs in aquatic crustaceans|
|关键词||Cpg Odns 甲壳动物 Toll 样受体 细胞免疫 体液免疫|
|其他摘要|| 水产养殖动物病害的频繁发生，已成为制约我国水产养殖可持续发展的主要瓶颈之一。环境友好型免疫增强剂的开发日渐成为领域内研究热点，其中CpG ODNs（Oligodeoxynucleotides）因其良好的抗菌、抗病毒效果而受到广泛关注。本研究以经济甲壳动物凡纳滨对虾（Litopenaeus vannamei）和中华绒螯蟹（Eriocheir sinensis）为研究对象，运用分子生物学及细胞生物学的相关手段，研究了不同类型CpG ODNs的免疫识别及其激活的下游免疫应答反应，明确了其抗病毒免疫效果及作用机制，为CpG ODNs在甲壳动物养殖中的应用提供理论依据。|
分析比较了四种不同类型CpG ODNs（CpG-A，CpG-B，CpG-C和CpG-P）的抗病毒效果。不同类型CpG ODNs处理凡纳滨对虾12小时后注射WSSV检测对虾鳃中的病毒拷贝数，发现注射WSSV后第五天，四种类型CpG ODNs处理组对虾鳃中病毒载量分别是对照组的18.19%, 48.82%, 57.75%和47.65%，抑制病毒的效果依次为CpG-A＞CpG-P＞CpG-B＞CpG-C，且CpG-A组的抑制效果与其余三组有显著差异。说明四种类型CpG ODNs均能够在不同程度上抑制凡纳滨对虾体内病毒拷贝数，且CpG-A具有相对较强的抗病毒免疫效果，可作为免疫增强剂应用于养殖对虾的抗病毒免疫防治中。
运用体内刺激实验和体外表面等离子体共振（SPR）技术研究LvTolls （LvToll1、LvToll2、LvToll3）对不同类型CpG ODNs的免疫识别活性。发现不同CpG处理后三种LvTolls的mRNA表达水平均有升高。与其他三种CpG ODNs处理及对照组相比，凡纳滨对虾肝胰腺中三种LvTolls的mRNA表达量在CpG-A处理后24小时显著升高，且LvToll1的mRNA表达水平显著高于LvToll2和LvToll3。CpG-P组凡纳滨对虾肝胰腺中LvTolls的表达也显著升高，而CpG-B和CpG-C处理组的升高幅度相对较小，且上调幅度均低于CpG-A处理组。在体外实验中，三种LvToll胞外段重组蛋白（LvToll-ECDs）具有显著的体外结合CpG-A的特性，该结合活性显著高于其他类型CpG ODNs。且LvToll1-ECD与CpG-A的结合活性均显著高于其他两种LvToll-ECDs与CpG-A的结合活性。以上结果表明CpG-A是激活凡纳滨对虾Toll通路的重要CpG 类型，且推测LvToll1是CpG-A在凡纳滨对虾体内的主要受体。
探讨了四种不同类型CpG ODNs诱导的下游免疫应答。发现CpG-A和CpG-B处理后凡纳滨对虾血细胞的吞噬能力和ROS水平显著升高，表明CpG-A和CpG-B能够激活凡纳滨对虾的细胞免疫应答；检测了对虾IRF-Vago-JAK/STAT通路相关指标，发现CpG-A处理后，对虾肝胰腺中LvIRF、LvVago4、LvVago5和LvSTAT的mRNA表达水平显著升高，血浆中类干扰素（IFN）含量显著增加，表明CpG-A能够激活对虾IFN system-like抗病毒系统，在对虾抗病毒免疫中起到重要作用。此外，CpG-B能够提高血浆中肿瘤坏死因子（TNF）的含量，CpG-C能够提高血浆中白细胞介素（IL）的含量，CpG-A和CpG-P能够提高血浆中类IFN的含量，与其在高等动物中的作用类似，表明不同类型CpG ODNs在凡纳滨对虾中的免疫激活作用存在差异。
利用RNA干扰技术干扰EsMyD88表达后检测CpG ODNs相关免疫指标的变化，探究CpG ODNs激活甲壳动物免疫反应的机制。结果显示，混合型CpG ODNs可以显著诱导中华绒螯蟹免疫相关基因EsProPO、CrusEs2、EsCAM、EsLITAF和EsmtMnSOD的mRNA表达，但是在干扰EsMyD88和CpG ODNs共处理后，这些基因的表达水平较CpG ODNs单独处理组显著降低，同时血浆中类IFN、IL和过氧化氢酶（CAT）的含量也显著低于CpG ODNs单独处理组的水平。推测CpG ODNs对中华绒螯蟹的部分免疫激活作用是由EsMyD88介导的。
综上所述，作为一种新型的免疫增强剂，CpG ODNs可以通过Toll通路激活甲壳动物固有免疫系统，诱导细胞免疫和体液免疫进而抵御病原入侵；不同类型CpG ODNs的免疫激活作用存在差异，其中A型最好，研究结果为其在水产养殖中的应用提供理论依据。
; The frequent outbreak of diseases in aquaculture animals produces harmful effect on aquaculture in China. The development of environmentally friendly immune substance has received great attention in aquaculture, among which, CpG ODNs (Oligodeoxynucleotides) have received extensive attention due to their great antibacterial and antiviral effects. In the present study, the recognition effect and the immune responses of different CpG ODNs in Litopenaeus vannamei and Eriocheir sinensis were explored by means of molecular biology and cell biology techniques, and their antiviral effect and related mechanism were further investigated, providing a theoretical basis for the application of CpG ODNs in aquaculture.
Four types of CpG ODNs (CpG-A, CpG-B, CpG-C and CpG-P) were selected to stimulate shrimps, and then shrimps were challenged with WSSV to detect the virus copy numbers in their gill tissue. The virus copy numbers after five days post injection in CpG-A, CpG-B, CpG-C and CpG-P group were 18.19%, 48.82%, 57.75% and 47.65% of that in PBS group, respectively. It showed that four types of CpG ODNs could inhibit WSSV copy numbers in different degree (CpG-A > CpG-P > CpG-B > CpG-C) and CpG-A played an important role in the process of antiviral immune response.
The immunological recognition activities of LvTolls (LvToll1, LvToll2, LvToll3) to different CpG ODNs and the mRNA expression levels of LvTolls after CpG ODNs stimulation were investigated. The mRNA expression level of three LvTolls in hepatopancreas increased significantly at 24 h post CpG-A stimulation. CpG-P could also significantly enhance the expression level of LvTolls, but the increase amplitude in CpG-B and CpG-C groups were relatively less. In addition, the mRNA expression level of LvToll1 was significantly higher than that of LvToll2 and LvToll3 in CpG-A group. In vitro experiment, CpG-A showed the strongest binding ability with three LvToll-ECDs. The binding ability of LvToll1-ECD with CpG-A was also significantly stronger than that of LvToll2-ECD and LvToll3-ECD with CpG-A, It indicated that CpG-A was an important type of CpG ODNs that could activate the Toll pathway in shrimp, and LvToll1 may be the main receptor of CpG-A in Litopenaeus vannamei.
The downstream immune responses induced by the four different types of CpG ODNs were explored. CpG-A and CpG-B could enhance phagocytosis and ROS levels of hemocytes，inducing cellular immune response. Relevant indicators in the IRF-Vago-JAK/STAT pathway were tested in shrimp. The expression levels of LvIRF, LvVago4, LvVago5 and LvSTAT were significantly up-regulated after CpG-A treatment, and the IFN content in plasma also increased, showing that CpG-A could activate the IFN system-like antiviral system, playing an important role in shrimp antiviral immunity. In addition, CpG-B could improve the content of TNF in plasma; CpG-C could increase the plasma levels of IL; CpG-A and CpG-P could improve the plasma level of IFN, revealed that different types of CpG ODNs showed different effects in shrimps, which were similar to their roles in mammals.
CpG ODNs related immunity indicators were detected after RNA interference of EsMyD88, to explore the mechanism of the immune responses activated by CpG ODNs in crustaceans. Results showed that mixed CpG ODNs could improve the mRNA expression levels of several immune related genes (EsProPO, CrusEs2, EsCAM, EsLITAF and EsmtMnSOD). But compared with the CpG ODNs treatment group, the expression levels of these genes in co-treatment of EsMyD88 interference and CpG ODNs group were significantly reduced. Moreover, the increase of IFN, IL and CAT(catalase) activity in plasma caused by CpG ODNs could also be reduced by EsMyD88 interference. The above results indicated that part of the immune functions induced by CpG ODNs in Eriocheir sinensis were mediated by EsMyD88.
In conclusion, as a novel immunostimulant, CpG ODNs could trigger the immune responses and induce great antiviral effect in crustaceans through the activation of Toll pathway. And different types of CpG ODNs showed diverse effects, providing a theoretical basis for their application in aquaculture.
|徐建超. 四种类型CpG ODNs 对水产养殖甲壳动物免疫调节作用的初步研究[D]. 北京. 中国科学院大学,2017.|
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