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Volume 46 Issue 1
Jan.  2022
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Role of tissue inhibitor of metalloproteinase in the anti-Vibrio immunity of Haliotis discus hannai

  • Corresponding author: CHEN Yulei, ylchen@jmu.edu.cn ; CAO Minjie, mjcao@jmu.edu.cn
  • Received Date: 2020-10-04
    Accepted Date: 2021-01-27
    Available Online: 2021-09-24
  • As an important economic shellfish in coastal area of China, abalone is susceptible to bacterial infection, especially Vibiro parahaemolyticus. Similar to other invertebrates, Haliotis discus hannai resists pathogen infection by innate immune response. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous protein regulators of the matrix metalloproteinases (MMPs) family. As MMP-1 was engaged in the innate immunity of abalone, the involvement of TIMP in the regulation of MMP-1 expression and activity as well as the immune responses merits further investigation. In this study, the role of TIMP in the innate immune responses of H. discus hannai against V. parahaemolyticus infection and the interaction between TIMP and MMP-1 were investigated. The full length cDNA sequence of TIMP was obtained, and the expressions of TIMP in different tissues of H. discus hannai were analyzed post Vibrio infection. The cloned TIMP cDNA sequence was 2291 bp in length. NetNGlyc 1.0 Server and Netoglyc 4.0 Server were used to analyze the glycosylation sites of TIMP. The results showed that TIMP had three potential N-glycosylation sites, namely Asn at positions 47, 77 and 152, and a potential O-glycosylation site, namely Thr at position 108. Multiple alignment of TIMP amino acid sequences from different molluscs showed that the similarities of TIMP in H. discus hannai with those of H. diversicolor, Crassostrea gigas and Tegillarca granosa were 76%, 18.9% and 19.3%, respectively. In the early stage of V. parahaemolyticus infection, the expression of TIMP in hemocytes and gill tissues were significantly up-regulated. To study the interaction between TIMP and MMP-1 in the anti-Vibrio immunity of abalone, RNA interference technology was used to knock down the expression levels of TIMP and MMP-1. After the silencing of MMP-1 gene in abalone, TIMP expression was significantly up-regulated. When TIMP expression was inhibited, MMP-1 expression decreased significantly. These results indicated that MMP-1 expression was positively regulated by TIMP, while TIMP expression was negatively regulated by MMP-1 in abalone. In conclusion, the present study will help to reveal the interaction between TIMP and MMP-1 in abalone innate immunity against V. parahaemolyticus infection. It also provided new insights into the prevention and control of pathogens in abalone culture.
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    [19] 牛海玲. 刺参MMP及TIMP基因的克隆及在自溶过程中的变化研究[D]. 大连: 大连工业大学, 2014.Niu H L. Molecular cloning of MMP and TIMP from Stichopus japonicus and their expression profile during autolysis[D]. Dalian: Dalian Polytechnic University, 2014 (in Chinese).
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    [24] Kübler A, Luna B, Larsson C, et al. Mycobacterium tuberculosis dysregulates MMP/TIMP balance to drive rapid cavitation and unrestrained bacterial proliferation[J]. Journal of Pathology, 2015, 235(3): 431-444. doi: 10.1002/path.4432
    [25] Brew K, Nagase H. The Tissue Inhibitors of Metalloproteinases (TIMPs): An ancient family with structural and functional diversity[J]. Biochimica et Biophysica Acta (BBA)- Molecular Cell Research, 2010, 1803(1): 55-71. doi: 10.1016/j.bbamcr.2010.01.003
    [26] 费晨. 基质金属蛋白酶在痘病毒感染中的作用和表达变化[D]. 杨凌: 西北农林科技大学, 2010.Fei C. Roles of matrix metalloproteinases in poxvirus replication[D]. Yangling: Northwest A & F University, 2010 (in Chinese).
    [27] Wang Q, Bao Y B, Huo L H, et al. A novel tissue inhibitor of metalloproteinase in blood clam Tegillarca granosa: molecular cloning, tissue distribution and expression analysis[J]. Fish & Shellfish Immunology, 2012, 33(3): 645-651.
    [28] Montagnani C, Le Roux F, Berthe F, et al. Cg-TIMP, an inducible tissue inhibitor of metalloproteinase from the pacific oyster Crassostrea gigas with a potential role in wound healing and defense mechanisms[J]. FEBS Letters, 2001, 500(1-2): 64-70. doi: 10.1016/S0014-5793(01)02559-5
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Role of tissue inhibitor of metalloproteinase in the anti-Vibrio immunity of Haliotis discus hannai

    Corresponding author: CHEN Yulei, ylchen@jmu.edu.cn
    Corresponding author: CAO Minjie, mjcao@jmu.edu.cn
  • 1. College of Ocean Food and Biological Engineering, Jimei University, Xiamen    361021, China
  • 2. National & Local Joint Engineering Research Center of Deep Processing Technology for Aquatic Products, Xiamen 361021, China

Abstract: As an important economic shellfish in coastal area of China, abalone is susceptible to bacterial infection, especially Vibiro parahaemolyticus. Similar to other invertebrates, Haliotis discus hannai resists pathogen infection by innate immune response. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous protein regulators of the matrix metalloproteinases (MMPs) family. As MMP-1 was engaged in the innate immunity of abalone, the involvement of TIMP in the regulation of MMP-1 expression and activity as well as the immune responses merits further investigation. In this study, the role of TIMP in the innate immune responses of H. discus hannai against V. parahaemolyticus infection and the interaction between TIMP and MMP-1 were investigated. The full length cDNA sequence of TIMP was obtained, and the expressions of TIMP in different tissues of H. discus hannai were analyzed post Vibrio infection. The cloned TIMP cDNA sequence was 2291 bp in length. NetNGlyc 1.0 Server and Netoglyc 4.0 Server were used to analyze the glycosylation sites of TIMP. The results showed that TIMP had three potential N-glycosylation sites, namely Asn at positions 47, 77 and 152, and a potential O-glycosylation site, namely Thr at position 108. Multiple alignment of TIMP amino acid sequences from different molluscs showed that the similarities of TIMP in H. discus hannai with those of H. diversicolor, Crassostrea gigas and Tegillarca granosa were 76%, 18.9% and 19.3%, respectively. In the early stage of V. parahaemolyticus infection, the expression of TIMP in hemocytes and gill tissues were significantly up-regulated. To study the interaction between TIMP and MMP-1 in the anti-Vibrio immunity of abalone, RNA interference technology was used to knock down the expression levels of TIMP and MMP-1. After the silencing of MMP-1 gene in abalone, TIMP expression was significantly up-regulated. When TIMP expression was inhibited, MMP-1 expression decreased significantly. These results indicated that MMP-1 expression was positively regulated by TIMP, while TIMP expression was negatively regulated by MMP-1 in abalone. In conclusion, the present study will help to reveal the interaction between TIMP and MMP-1 in abalone innate immunity against V. parahaemolyticus infection. It also provided new insights into the prevention and control of pathogens in abalone culture.

  • 鲍隶属于软体动物门(Mollusca)腹足纲(Gastropoda)原始腹足目(Archaeogastropoda)鲍科(Haliotidae)鲍属(Haliotis),具有较高的营养和药学价值,位列“海产八珍”之一,是非常重要的渔业资源和水产养殖品种[1]。2019年我国鲍产量近18万t,较2018年同比增长10.48%,福建省鲍的产量占全国80%,位居首位[2]。皱纹盘鲍(Haliotis discus hannai)原为我国北部沿海重要的渔获种类,也是该地区最优良的浅海养殖贝类之一[3]。由于福建等南方海域冬季水温较高、皱纹盘鲍生长速率快,“北鲍南养”养殖方式得到很好地实施[4]。这不仅提高了鲍鱼的成活率,而且降低了养殖成本,鲍鱼的价格也逐渐被普通消费者所接受。

    由弧菌感染引起的疾病是影响鲍鱼产量和质量的主要因素之一[5]。其中,副溶血性弧菌(Vibrio parahaemolyticus)的致死率最高[6]。该菌是一种嗜盐性的革兰氏阴性菌,可以附着在海洋生物体内,对海洋生物和人类都具有很强的致病性[7]。与其他无脊椎动物类似,皱纹盘鲍利用先天性免疫反应来抵御病原菌对宿主的侵染。皱纹盘鲍的先天性免疫系统包含多种受体、调控因子和效应蛋白,例如纤维蛋白原相关蛋白、Toll样受体、清道夫受体和抗菌肽等[8]。除此之外,参与血淋巴凝集、血淋巴细胞吞噬和凋亡等主要过程的大分子也在皱纹盘鲍的先天性免疫过程中发挥重要作用[9]

    基质金属蛋白酶(matrix metalloproteinases,MMPs)是一类高度保守的依赖于锌离子和钙离子的蛋白水解酶[10]。MMPs的表达由糖皮质激素、类维生素A、病菌感染和外界压力等因素调控[11],其活性受转录水平、酶原激活和金属蛋白酶组织抑制剂(tissue inhibitors of MMPs,TIMPs)的严格控制[12]。TIMPs 是一组低分子量的糖蛋白,广泛分布于组织和体液中,可由成纤维细胞、上皮细胞、内皮细胞等产生[13]。作为组织中MMPs主要的内源性抑制因子,TIMPs能特异性抑制MMPs家族的基质降解酶。TIMPs与不同的MMPs表现出不同的亲和力,其以可逆的共价键方式与MMPs相结合形成1∶1复合体,二者的平衡关系在调节胞外基质(extracellular matrix,ECM)的稳定中有重要作用[14]。作为多功能分子,TIMPs不仅可抑制MMPs活性,且具有细胞生长因子样作用,可促进成纤维细胞增生及胶原合成,使ECM沉积。此外,TIMPs还可调控ECM代谢和抑制血管的生成,抑制肿瘤发生和发展[15]

    Chen等[16]研究表明,副溶血性弧菌感染导致皱纹盘鲍MMP-1表达量显著上调,且MMP-1可调节免疫相关基因的表达及细胞的活性,但其内源性抑制剂TIMP的具体功能尚不清楚。因此,本实验以皱纹盘鲍为研究对象,克隆得到TIMP序列,分析了TIMP在皱纹盘鲍不同组织及副溶血性弧菌感染前后的表达情况,并初步探索了TIMP与MMP-1的相互作用关系。本研究有助于了解皱纹盘鲍抗弧菌感染的分子机理,对了解TIMPs与MMPs在软体动物免疫中的相互作用具有重要意义。

1.   材料与方法
  • 大小均一的健康皱纹盘鲍购于福建省厦门市夏商国际水产交易中心,平均体质量约40 g,于20 °C暂养3~4 d后根据相关实验分组进行处理。养殖用海水取自集美大学水产试验场,每12 h更换1次。

  • 实验用皱纹盘鲍暂养后,随机挑取3只,收集血淋巴细胞、肝胰腺、鳃、肌肉、褶边、外套膜和性腺,不同个体相同组织混合后置于液氮备用。将剩余皱纹盘鲍随机分为2组,每组20只,试验组注射副溶血弧菌(2×107 CFU),对照组注射同等体积PBS。注射12、24、48和72 h后,分别随机挑取3只,收集皱纹盘鲍主要免疫器官(血淋巴细胞、鳃、肝胰腺),不同个体相同组织混合后置于液氮备用。以上实验均重复3次。

  • 按总RNA提取试剂盒(Tiangen)提供的方法提取皱纹盘鲍各组织(血淋巴细胞、肝胰腺、鳃、肌肉、褶边、外套膜和性腺)的总RNA。通过1.5%的琼脂糖凝胶电泳检测产物的完整性,使用Nanodrop ND1000检测RNA浓度及纯度。按PrimeScriptTM Ⅱ 1st Strand cDNA Synthesis Kit (TAKARA)操作说明合成普通克隆用cDNA。利用SMARTer RACE 5′/3′ Kit(TAKARA)合成cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)用cDNA。将不同组织mRNA浓度调为一致,利用ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO)合成用于荧光定量PCR (quantitative real-time PCR,qPCR)的cDNA。

  • 根据杂色鲍(H. diversicolor) TIMP序列设计正反向引物(表1),以皱纹盘鲍cDNA为模板扩增TIMP开放阅读框(open reading frame,ORF)序列。根据克隆得到的ORF序列设计RACE扩增用正反向引物(表1),以5′, 3′-RACE cDNA为模板,扩增5′和3′非编码区(untranslated region, UTR),最终得到皱纹盘鲍TIMP序列全长。

    引物名称
    primer name
    引物序列(5′-3′)
    primer sequence
    TIMP (ORF)-F ATGGAGTTTTCACCAGCTGTTTTG
    TIMP (ORF)-R TCAACATGTCAGGTTGGGAAAGTT
    TIMP-5′ RACE GGCGCAAAGTCGGCTTGACAGAAGTA
    TIMP-3′ RACE TCACATTGCCCTGTCATGGAGATGGA

    Table 1.  Primers used for TIMP cloning

  • 利用NetNGlyc 1.0 Server和NetOGlyc 4.0 Server软件分别对TIMP蛋白序列中潜在的N-糖基化位点和O-糖基化位点进行预测。在NCBI中用BLAST工具(http://blast.ncbi.nlm.nih.gov/Blast.cgi) 搜索得到皱纹盘鲍TIMP同源蛋白序列,利用DNAMAN软件进行多序列比对,通过Mega 7.0软件构建不同物种TIMP氨基酸序列的系统进化树。

  • 根据皱纹盘鲍TIMPMMP-1基因序列设计qPCR用引物(表2),以β-actin作为内参基因,利用TransStart® Top Green qPCR SuperMix (TransGen Biotech)分析TIMPMMP-1在皱纹盘鲍各组织及副溶血性弧菌感染前后的表达差异性。qPCR反应体系(10 μL):2×qPCR SuperMix 5 μL,正向引物(10 μmol/L) 0.2 μL,反向引物(10 μmol/L)0.2 μL,50×Passive Reference Dye 0.2 μL,cDNA 1.0 μL,ddH2O 3.4 μL。于ABI 7300仪器中进行两步法PCR扩增:94 °C预变性30 s;94 °C变性5 s,60 °C延伸31 s (荧光采集),循环40次;扩增结束后,从55 °C升温至94 °C制备熔解曲线。每个样品设置3个重复,每组反应均设置无模板对照。

    基因名称
    gene name
    正向引物(5′-3′)
    forward primer
    反向引物(5′-3′)
    reverse primer
    TIMP TTCGTCTTTGAGGCTTTGGTA ATCACAGGAGAAGTCAGGGAA
    MMP-1 GGAACACGGAGATGGACAATAC GTTGGGGGGTGAGGATTAGAC
    β-actin AGATGTTGCTGCGTTGGTTAT GATGGGGTACTTGAGGGTGAG

    Table 2.  Primers used in the qPCR assay

  • 利用siRNA在线设计网站(https://rnaidesigner.thermofisher.com/rnai-express)分别设计3条皱纹盘鲍TIMPMMP-1特异性siRNA,选择打乱序列的siRNA作为对照,siRNA序列见表3。利用In vitro transcription T7 kit (TaKaRa)体外合成成熟序列siRNA,将其溶解于siRNA溶液(50 mmol/L Tris-HCl,100 mmol/L NaCl,pH 7.5)中,并用Nanodrop ND-1000测定浓度。将副溶血性弧菌悬液和30 μg特异性的siRNA或打乱序列的siRNA共同注射至皱纹盘鲍肌肉内。此外,将只注射PBS的溶液设置为空白对照组,只注射副溶血性弧菌溶液设置为阴性对照组。每组注射12、24、48和72 h后,收集3只皱纹盘鲍的血淋巴液,于4 °C 500×g离心10 min,离心收集的血淋巴细胞用于后续检测。以上实验重复3次。

    siRNA序列名称
    sequence name
    正义链 (5′-3′)
    sense strand
    反义链 (5′-3′)
    anti-sense strand
    TIMP-siRNA1 CCAACTGGACCACTGACAA TTGTCAGTGGTCCAGTTGG
    TIMP-siRNA2 CCCTGTCATGGAGATGGAT
    ATCCATCTCCATGACAGGG
    TIMP-siRNA3 GGACATACTTGCTTACCAT ATGGTAAGCAAGTATGTCC
    MMP1-siRNA1 GCTACCTCAACACCGACTA TAGTCGGTGTTGAGGTAGC
    MMP1-siRNA2 GGTTGCTGCTCATGAATTT AAATTCATGAGCAGCAACC
    MMP1-siRNA3 GCAAGAGCCGCATCTTCAT ATGAAGATGCGGCTCTTGC
    scrambled siRNA TTCTCCGAACGTGTCACGT ACGTGACACGTTCGGAGAA

    Table 3.  siRNAs used in the RNA interference assay

  • 运用GraphPad Prism 5软件处理数据及图表绘制,采用单因素方差分析法(One-Way ANOVA)进行统计分析,显著性水平为P<0.05,极显著性水平为P<0.01。

2.   结果
  • 皱纹盘鲍肌肉组织中胶原蛋白含量高[3],富含多种与肌肉蛋白新陈代谢密切相关的金属蛋白酶。转录组测序结果显示,当弧菌感染杂色鲍后,其血淋巴细胞中的TIMPMMP-1表达量均发生显著上调[17]。作为MMPs的天然抑制剂,TIMPs极有可能通过调控MMPs的表达与活性来参与皱纹盘鲍的抗弧菌免疫过程。但目前关于TIMPs在鲍鱼抗菌免疫中的作用及其与MMPs的相互作用关系却知之甚少。

  • 以皱纹盘鲍血淋巴细胞cDNA为模板,克隆得到TIMP cDNA全长序列(图1)。该cDNA全长为2291 bp,其中5′非编码区(untranslated region,UTR) 133 bp,3′-UTR 1690 bp,开放阅读框(open reading frame,ORF) 468 bp,共编码155个氨基酸残基。推导的氨基酸序列包含一个N端信号肽(1~22 aa)和纺锤蛋白(nutrin,NTR)结构域,NTR结构域中有6个保守的半胱氨酸残基。利用NetNGlyc 1.0 Server对TIMP的氨基酸序列进行分析,结果显示其含有3个潜在的N-糖基化位点,分别是位于第47、77和152位的Asn;使用NetOGlyc 4.0 Server软件对TIMP的氨基酸序列进行分析,得到其含有一个潜在的O-糖基化位点,是位于第108位的Thr。

    Figure 1.  Nucleotide sequence and deduced amino acid sequence of TIMP

    使用DNAMAN软件对不同物种TIMP氨基酸序列进行多序列比对,结果显示,皱纹盘鲍TIMP氨基酸序列与杂色鲍、长牡蛎(Crassostrea gigas)、泥蚶(Tegillarca granosa)中TIMP的序列相似性分别为76.0%、18.9%和19.3% (图2),表明TIMP在软体动物中的保守性较低。

    Figure 2.  Multiple alignment of TIMP amino acid sequences from different molluscs

    通过Mega 7.0软件构建不同物种TIMP氨基酸序列的系统进化树。该系统进化树采用邻接法构建得到,Bootstrap值为1000,酿酒酵母属真菌界,因此选取酿酒酵母(Saccharomyces cerevisiae)作为有根树的外群。同为鲍属的皱纹盘鲍和杂色鲍形成姐妹群,支持率较高(自展值为100),在该分支之后又形成2个分支,分别是与同属软体动物门的长牡蛎和泥蚶,表明皱纹盘鲍的TIMP在进化分支上更接近软体动物,表现出与软体动物TIMP的共同祖先具有更高的同源性,而与其他远缘关系的物种表现出较大的差异 (图3)。综上所述,不同物种的TIMP在进化树上表现出较大的差异,关于脊索动物门(Chordata)中TIMP的研究已有较多报道,而软体动物中TIMP的研究目前报道较少,有待进一步研究。

    Figure 3.  Phylogenetic tree of TIMP from different species based on Neighbor-joining method

  • 利用荧光定量PCR技术检测皱纹盘鲍各组织中TIMP的表达情况。结果显示,TIMP在皱纹盘鲍各组织中均有表达(图4)。在转录水平上,TIMP在血淋巴细胞中表达量最高,其次为褶边和外套膜,而性腺、鳃、肌肉和肝胰腺中TIMP相对表达量较低(图4)。

    Figure 4.  Expression of TIMP in abalone tissues and organs

  • 为了进一步探究TIMP与皱纹盘鲍抗菌免疫的关系,首先利用副溶血性弧菌感染鲍,并收集不同感染时期的皱纹盘鲍免疫器官和组织,以检测TIMP表达与副溶血性弧菌感染的关系。结果显示,与PBS组相比较,副溶血性弧菌感染12 h时血淋巴细胞和鳃组织中TIMP转录水平升高了4倍,且均达到最高值。而后,TIMP转录水平显著下降至与对照组相近水平。弧菌感染并未引起肝胰腺中TIMP转录水平显著变化(图5)。

    Figure 5.  Expression of TIMP before and post V. parahaemolyticus infection

  • TIMPs是组织中MMPs主要的内源性抑制因子,能特异性抑制MMPs家族的基质降解酶。为了检测TIMP与MMP-1在皱纹盘鲍体内的相互作用关系,利用RNA干扰技术分别敲低皱纹盘鲍TIMPMMP-1表达量。当TIMP敲低24~72 h时,MMP-1表达量受到显著抑制(图6)。而当MMP-1敲低24 h后,TIMP表达量显著上调(图7)。结果表明,MMP-1表达受TIMP正调控,而TIMP表达受MMP-1负调控。

    Figure 6.  Effects of TIMP knock-down on the expression of MMP-1

    Figure 7.  Effects of MMP-1 knock-down on the expression of TIMP

3.   讨论
  • MMPs是参与降解ECM过程中至关重要的蛋白酶[18],TIMPs是该蛋白酶的内源性抑制剂[19],它同时也参与了细胞增殖、迁移、侵袭、血管生成抑制及细胞凋亡等进程[20]。在机体中,大部分情况下TIMPs都是通过抑制MMPs的活性来发挥作用[21],只有少部分情况TIMPs是通过与特殊的细胞受体相互结合发挥作用的。二者之间的动态平衡与体内细胞水平的信号传导、生长因子等息息相关。目前,软体动物中已有杂色鲍、长牡蛎、泥蚶等的TIMP序列被克隆,但对于TIMPs在软体动物先天性免疫中的研究及其与MMPs的相互作用关系鲜有报道。本研究根据杂色鲍TIMP序列设计引物,利用PCR技术克隆得到了皱纹盘鲍TIMP cDNA全长序列。该cDNA全长为2291 bp,其中ORF为468 bp,共编码155个氨基酸残基。推导的氨基酸序列包含一个N端信号肽(1~22 aa)和纺锤蛋白(nutrin,NTR)结构域,NTR结构域中有6个保守的半胱氨酸残基。

    已有研究表明,ECM成分是微生物病原体入侵的停靠点[22]。TIMPs对ECM的代谢至关重要,最主要的功能是直接抑制MMPs依赖的ECM蛋白水解作用,从而参与ECM的组织重排[23]。此外,MMPs和TIMPs参与多种生物活性蛋白的加工过程,包括细胞因子、趋化因子和细胞表面蛋白等,进而间接影响ECM的代谢[24]。破坏MMPs与TIMPs之间的平衡可能会导致与失控的细胞外基质周转、炎症、细胞生长和迁移相关的疾病,如关节炎、心血管疾病、癌症、肺病、肾炎、神经障碍和组织溃疡[25]。Chen等[16]研究表明,副溶血性弧菌感染导致皱纹盘鲍MMP-1表达量显著上调,且MMP-1可调节免疫相关基因的表达及细胞的活性。作为MMPs的内源性抑制剂,TIMPs极有可能通过调控MMPs的表达与活性来参与皱纹盘鲍的抗弧菌免疫过程。

    在水生无脊椎动物中,血淋巴细胞作为主要的免疫细胞,在抵御病菌感染过程中发挥重要作用[26]。转录组测序结果显示,当弧菌感染杂色鲍后,其血淋巴细胞中的TIMPMMP-1表达量均发生显著上调[17]。Wang等[27]对泥蚶血中的TIMP进行克隆表达,发现TIMP-4蛋白可能在软体动物防御机制中起着关键作用。Montagnani等[28]发现,TIMP与长牡蛎的个体发生、伤口愈合和防御机制密切相关。本研究结果表明,在弧菌感染早期,TIMP在血淋巴细胞和鳃组织中的表达量均显著上调,说明TIMP可能参与皱纹盘鲍的早期免疫应答反应。此外,皱纹盘鲍体内MMP-1表达受TIMP正调控,而TIMP表达受MMP-1负调控。以此推测,弧菌感染后皱纹盘鲍体内MMP-1表达量的升高可抑制TIMP的表达,从而形成负反馈调节。但弧菌感染诱导MMP-1和TIMP表达的先后顺序、MMP-1与TIMP蛋白水平的相互调控作用以及TIMP对MMP-1酶活性的抑制作用有待进一步探索。

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