本文主要研究内容
作者涂笑（2019）在《牛蒡子苷元作用鱼类单殖吸虫靶标及机制研究》一文中研究指出：单殖吸虫（Monogenean）是一类常见的鱼类寄生虫,广泛分布于世界各地淡、海水水域。常因大量寄生及其引起的继发性疾病感染,造成幼鱼大量死亡,给水产养殖业带来严重的经济损失。指环虫属（Dactylogyrus）和三代虫属（Gyrodactylus）是单殖吸虫纲中物种数目最丰富、也是危害最严重的属。目前,渔业生产中,国标渔药因长期、连年持续增加使用剂量引起寄生虫的耐药性发生,加大了单殖吸虫病防治难度,因此,新药的创制引起水产领域极大的关注。天然产物不仅生物活性丰富而且骨架结构多样,是药物研发的理想来源。前期,本实验室运用活性跟踪法从药用植物中分离鉴定出多种杀灭指环虫的活性小分子,其中牛蒡子苷元（Arctigenin,ARG）杀虫效果显著、对鱼类毒性低,是理想的开发成为专用杀虫渔药的先导化合物。然而,缺乏对先导化合物作用靶点和机制的研究将限制先导化合物的进一步结构优化,阻碍药物的研发进程。本研究首先构建小林三代虫（Gyrodactylus kobayashii）感染金鱼实验模型,在此基础上,通过ARG作用小林三代虫形态学、RNA-seq转录组学和iTRAQ定量蛋白组学以及利用金纳米探针分离鉴定ARG作用靶点蛋白,揭示ARG杀虫机制。取得结果如下:1.小林三代虫感染金鱼实验模型的建立采用分子生物学鉴定方法确定自然感染金鱼的三代虫种类中小林三代虫（G.kobayashii）是绝对优势种（70.8%）;随后,通过吡喹酮药浴获得无虫金鱼、尾鳍人工接种感染、三代虫种类鉴定和种群维持等步骤,建立了小林三代虫感染金鱼系统。采用ApoⅠ酶切法定期对系统中三代虫种类进行鉴定,结果发现:随机采样获得的144条（12条/次）三代虫均属于小林三代虫,表明该实验模型三代虫种群单一且能稳定传代。最后,利用该模型研究三代虫感染规律及宿主免疫反应,结果显示小林三代虫种群数量在感染前8天呈爆发式增长,随后急剧下降并保持低密度感染;金鱼肝脾肾组织中促炎性细胞因子如TNF-α、IL-1β、IFN-γ以及iNOS等在整个三代虫感染过程中表达量均显著上调,其中iNOS表达量最高,同样,在感染三代虫的金鱼血清中一氧化氮的含量也发现显著升高,说明细胞因子的表达在清除三代虫感染中起着重要作用。以上结果表明:定期添加无虫金鱼是获得大量小林三代虫的关键,为下一步药物作用靶点和机制研究奠定基础。2.牛蒡子苷元安全性评价及作用小林三代虫形态学研究利用上述构建的实验模型进行ARG杀虫活性及安全性评价,结果显示:离体条件下,8 mg/L ARG在33 min内即可造成小林三代虫全部死亡;在体条件下,4.00 mg/L ARG作用4 h即可达100%杀虫率,24 h和48 h的EC50分别为1.85 mg/L和1.58mg/L。ARG对金鱼安全性评价结果发现:96 h对金鱼的LC50值为11.63 mg/L（95%置信区间为11.29-11.99 mg/L）;1.85 mg/L(EC50)的ARG浸泡24 h引起金鱼肝脏外源物质敏感基因（cyp1a、hsp70、gst及sod）差异表达,48 h和96 h时表达量与对照组差异不显著（p<0.05）;4 mg/L(EC99)的ARG浸泡96 h sod的mRNA水平与初始水平无显著性差异,其余3个基因表达量呈明显的恢复趋势,表明ARG对金鱼毒性较低。ARG药浴后可见三代虫的活动能力急剧下降,而后直接中毒死亡,与呼吸抑制剂鱼藤酮和寡霉素A引起中毒症状类似;扫描电镜观察三代虫表皮出现明显的损伤;透射电镜观察发现三代虫表皮肌肉结构出现明显的溶解,线粒体可见大规模肿胀和空泡化现象;肌动蛋白荧光检测结果显示三代虫肌肉结构明显被破坏。以上结果表明ARG作用三代虫靶点可能位于表皮肌肉结构或者线粒体中,杀虫机制可能与表皮损伤以及呼吸被抑制有关。3.牛蒡子苷元对小林三代虫转录组水平的影响采用转录组测序（RNA-seq）技术研究了ARG作用对小林三代虫转录组水平的影响,筛选相关差异基因并进行生物信息学分析,探讨ARG杀虫分子机制和潜在作用靶点。结果发现:（1）与空白对照组相比,4 mg/L ARG,0.5 h处理组共获234个差异表达基因;1.85 mg/L ARG,0.5 h处理组共获118个差异表达基因;1.85 mg/L ARG,4h处理组共获4936个差异表达基因,其中上调755个,下调4181个。（2）GO注释结果表明差异表达基因主要参与胞外基质组成（extracellular matrix）、电子传递链（electron carrier activity）、大分子跨膜活性（macromolecule transmembrane transporter activity）以及能量代谢过程（energy metabolic process）等重要的机体过程。（3）KEGG通路富集分析发现差异表达基因富集在蛋白质加工、细胞外基质相互作用、糖酵解/糖异生过程、心肌收缩,氧化磷酸化等途径。（4）以上结果表明ARG主要通过破坏三代虫表皮正常生理结构,抑制能量代谢通路中某些关键基因的表达,从而造成虫体死亡。4.牛蒡子苷元作用小林三代虫蛋白质组比较研究采用iTRAQ定量蛋白组学技术研究ARG对小林三代虫蛋白质表达谱的影响,对差异表达的蛋白质进行GO功能注释、KEGG通路富集分析,并结合转录组学数据探讨ARG作用三代虫潜在作用靶点和分子机制。结果显示:（1）共鉴定到总蛋白2850个;与空白对照组相比,4 mg/L,0.5 h处理组、1.85 mg/L 0.5 h和4 h处理组分别引起335个、223个和313个差异表达蛋白,三个ARG处理组中均差异表达蛋白82个。（2）生物信息学分析结果显示差异表达蛋白主要参与细胞骨架、离子转运以及肌肉收缩等生理过程。（3）转录组学和蛋白质组学关联分析发现19个mRNA和蛋白水平差异表达一致的蛋白,也是ARG直接或间接的药物杀虫靶点,被富集到肌动蛋白细胞骨架调节、氧化磷酸化（oxidative phosphorylation）、内吞作用（Endocytosis）以及黏着斑（Focal adhesion）等通路中。其中ATP synthase E chain（ATP合成酶E链）参与机体ATP直接合成过程。（4）进一步利用qRT-PCR技术研究ARG对三代虫ATP合成酶不同亚基以及呼吸链复合物Ⅰ-Ⅳ基因表达的影响,结果发现各基因经1.85 mg/L的ARG处理6、12和24 h后均显著下调;此外经ARG离体和在体处理后的三代虫体内ATP含量均急剧降低,表明ARG确实能引起三代虫能量供应不足。5.利用金纳米探针鉴定牛蒡子苷元作用小林三代虫靶点利用金纳米探针法通过亲和层析原理直接从三代虫的总蛋白中直接分离ARG作用小林三代虫靶点蛋白,取得的结果如下:（1）首先通过破坏ARG内酯环获得化合物2,其结构与ARG类似但活性差异巨大(EC50为68.9 mg/L),选为阴性对照排除假阳性蛋白;（2）ARG和化合物2分别进行侧链羟基修饰连接硫辛酸结构,使其通过金硫键连接到金纳米颗粒表面,获得金纳米探针GNP-6（ARG连接的金纳米颗粒）和GNP-10（化合物2连接的金纳米颗粒）;（3）高分辨透射电镜观察到金纳米探针GNP-6呈均匀的球形,平均粒径为3.58 nm,此外,还可观察到GNP-6能进入小林三代虫细胞核等部位,且仍保持杀灭小林三代虫活性;（4）将GNP-6和GNP-10分别与小林三代虫蛋白裂解液孵育,经SDS-PAGE分离,获得两条GNP-6中特异性条带,通过基质辅助激光解析电离飞行时间质谱（MALDI TOF/TOF MS）分析并采用Mascot 2.2软件搜索Uniprot秀丽隐杆线虫数据库,确定了ARG的靶点蛋白为肌球蛋白（MyosinⅡ）和肌肉M线装配蛋白（Muscle M-line assembly protein,UNC-89）。综上所述,本研究基于构建的小林三代虫感染金鱼实验模型,进行形态学研究、转录组学和蛋白质学组关联分析以及靶点蛋白的分离鉴定,初步确定ARG杀灭鱼类单殖吸虫作用靶点是MyosinⅡ和UNC-89,均与维持肌球蛋白正常结构有关,表明ARG杀虫机制与药物破坏肌球蛋白结构,引起虫体能量供应不足最终死亡有关。上述结果为ARG进一步结构优化提供了重要参考资料。
Abstract
chan shi xi chong （Monogenean）shi yi lei chang jian de yu lei ji sheng chong ,an fan fen bu yu shi jie ge de dan 、hai shui shui yu 。chang yin da liang ji sheng ji ji yin qi de ji fa xing ji bing gan ran ,zao cheng you yu da liang si wang ,gei shui chan yang shi ye dai lai yan chong de jing ji sun shi 。zhi huan chong shu （Dactylogyrus）he san dai chong shu （Gyrodactylus）shi chan shi xi chong gang zhong wu chong shu mu zui feng fu 、ye shi wei hai zui yan chong de shu 。mu qian ,yu ye sheng chan zhong ,guo biao yu yao yin chang ji 、lian nian chi xu zeng jia shi yong ji liang yin qi ji sheng chong de nai yao xing fa sheng ,jia da le chan shi xi chong bing fang zhi nan du ,yin ci ,xin yao de chuang zhi yin qi shui chan ling yu ji da de guan zhu 。tian ran chan wu bu jin sheng wu huo xing feng fu er ju gu jia jie gou duo yang ,shi yao wu yan fa de li xiang lai yuan 。qian ji ,ben shi yan shi yun yong huo xing gen zong fa cong yao yong zhi wu zhong fen li jian ding chu duo chong sha mie zhi huan chong de huo xing xiao fen zi ,ji zhong niu bang zi gan yuan （Arctigenin,ARG）sha chong xiao guo xian zhe 、dui yu lei du xing di ,shi li xiang de kai fa cheng wei zhuan yong sha chong yu yao de xian dao hua ge wu 。ran er ,que fa dui xian dao hua ge wu zuo yong ba dian he ji zhi de yan jiu jiang xian zhi xian dao hua ge wu de jin yi bu jie gou you hua ,zu ai yao wu de yan fa jin cheng 。ben yan jiu shou xian gou jian xiao lin san dai chong （Gyrodactylus kobayashii）gan ran jin yu shi yan mo xing ,zai ci ji chu shang ,tong guo ARGzuo yong xiao lin san dai chong xing tai xue 、RNA-seqzhuai lu zu xue he iTRAQding liang dan bai zu xue yi ji li yong jin na mi tan zhen fen li jian ding ARGzuo yong ba dian dan bai ,jie shi ARGsha chong ji zhi 。qu de jie guo ru xia :1.xiao lin san dai chong gan ran jin yu shi yan mo xing de jian li cai yong fen zi sheng wu xue jian ding fang fa que ding zi ran gan ran jin yu de san dai chong chong lei zhong xiao lin san dai chong （G.kobayashii）shi jue dui you shi chong （70.8%）;sui hou ,tong guo bi kui tong yao yu huo de mo chong jin yu 、wei qi ren gong jie chong gan ran 、san dai chong chong lei jian ding he chong qun wei chi deng bu zhou ,jian li le xiao lin san dai chong gan ran jin yu ji tong 。cai yong ApoⅠmei qie fa ding ji dui ji tong zhong san dai chong chong lei jin hang jian ding ,jie guo fa xian :sui ji cai yang huo de de 144tiao （12tiao /ci ）san dai chong jun shu yu xiao lin san dai chong ,biao ming gai shi yan mo xing san dai chong chong qun chan yi ju neng wen ding chuan dai 。zui hou ,li yong gai mo xing yan jiu san dai chong gan ran gui lv ji su zhu mian yi fan ying ,jie guo xian shi xiao lin san dai chong chong qun shu liang zai gan ran qian 8tian cheng bao fa shi zeng chang ,sui hou ji ju xia jiang bing bao chi di mi du gan ran ;jin yu gan pi shen zu zhi zhong cu yan xing xi bao yin zi ru TNF-α、IL-1β、IFN-γyi ji iNOSdeng zai zheng ge san dai chong gan ran guo cheng zhong biao da liang jun xian zhe shang diao ,ji zhong iNOSbiao da liang zui gao ,tong yang ,zai gan ran san dai chong de jin yu xie qing zhong yi yang hua dan de han liang ye fa xian xian zhe sheng gao ,shui ming xi bao yin zi de biao da zai qing chu san dai chong gan ran zhong qi zhao chong yao zuo yong 。yi shang jie guo biao ming :ding ji tian jia mo chong jin yu shi huo de da liang xiao lin san dai chong de guan jian ,wei xia yi bu yao wu zuo yong ba dian he ji zhi yan jiu dian ding ji chu 。2.niu bang zi gan yuan an quan xing ping jia ji zuo yong xiao lin san dai chong xing tai xue yan jiu li yong shang shu gou jian de shi yan mo xing jin hang ARGsha chong huo xing ji an quan xing ping jia ,jie guo xian shi :li ti tiao jian xia ,8 mg/L ARGzai 33 minnei ji ke zao cheng xiao lin san dai chong quan bu si wang ;zai ti tiao jian xia ,4.00 mg/L ARGzuo yong 4 hji ke da 100%sha chong lv ,24 hhe 48 hde EC50fen bie wei 1.85 mg/Lhe 1.58mg/L。ARGdui jin yu an quan xing ping jia jie guo fa xian :96 hdui jin yu de LC50zhi wei 11.63 mg/L（95%zhi xin ou jian wei 11.29-11.99 mg/L）;1.85 mg/L(EC50)de ARGjin pao 24 hyin qi jin yu gan zang wai yuan wu zhi min gan ji yin （cyp1a、hsp70、gstji sod）cha yi biao da ,48 hhe 96 hshi biao da liang yu dui zhao zu cha yi bu xian zhe （p<0.05）;4 mg/L(EC99)de ARGjin pao 96 h sodde mRNAshui ping yu chu shi shui ping mo xian zhe xing cha yi ,ji yu 3ge ji yin biao da liang cheng ming xian de hui fu qu shi ,biao ming ARGdui jin yu du xing jiao di 。ARGyao yu hou ke jian san dai chong de huo dong neng li ji ju xia jiang ,er hou zhi jie zhong du si wang ,yu hu xi yi zhi ji yu teng tong he gua mei su Ayin qi zhong du zheng zhuang lei shi ;sao miao dian jing guan cha san dai chong biao pi chu xian ming xian de sun shang ;tou she dian jing guan cha fa xian san dai chong biao pi ji rou jie gou chu xian ming xian de rong jie ,xian li ti ke jian da gui mo zhong zhang he kong pao hua xian xiang ;ji dong dan bai ying guang jian ce jie guo xian shi san dai chong ji rou jie gou ming xian bei po huai 。yi shang jie guo biao ming ARGzuo yong san dai chong ba dian ke neng wei yu biao pi ji rou jie gou huo zhe xian li ti zhong ,sha chong ji zhi ke neng yu biao pi sun shang yi ji hu xi bei yi zhi you guan 。3.niu bang zi gan yuan dui xiao lin san dai chong zhuai lu zu shui ping de ying xiang cai yong zhuai lu zu ce xu （RNA-seq）ji shu yan jiu le ARGzuo yong dui xiao lin san dai chong zhuai lu zu shui ping de ying xiang ,shai shua xiang guan cha yi ji yin bing jin hang sheng wu xin xi xue fen xi ,tan tao ARGsha chong fen zi ji zhi he qian zai zuo yong ba dian 。jie guo fa xian :（1）yu kong bai dui zhao zu xiang bi ,4 mg/L ARG,0.5 hchu li zu gong huo 234ge cha yi biao da ji yin ;1.85 mg/L ARG,0.5 hchu li zu gong huo 118ge cha yi biao da ji yin ;1.85 mg/L ARG,4hchu li zu gong huo 4936ge cha yi biao da ji yin ,ji zhong shang diao 755ge ,xia diao 4181ge 。（2）GOzhu shi jie guo biao ming cha yi biao da ji yin zhu yao can yu bao wai ji zhi zu cheng （extracellular matrix）、dian zi chuan di lian （electron carrier activity）、da fen zi kua mo huo xing （macromolecule transmembrane transporter activity）yi ji neng liang dai xie guo cheng （energy metabolic process）deng chong yao de ji ti guo cheng 。（3）KEGGtong lu fu ji fen xi fa xian cha yi biao da ji yin fu ji zai dan bai zhi jia gong 、xi bao wai ji zhi xiang hu zuo yong 、tang jiao jie /tang yi sheng guo cheng 、xin ji shou su ,yang hua lin suan hua deng tu jing 。（4）yi shang jie guo biao ming ARGzhu yao tong guo po huai san dai chong biao pi zheng chang sheng li jie gou ,yi zhi neng liang dai xie tong lu zhong mou xie guan jian ji yin de biao da ,cong er zao cheng chong ti si wang 。4.niu bang zi gan yuan zuo yong xiao lin san dai chong dan bai zhi zu bi jiao yan jiu cai yong iTRAQding liang dan bai zu xue ji shu yan jiu ARGdui xiao lin san dai chong dan bai zhi biao da pu de ying xiang ,dui cha yi biao da de dan bai zhi jin hang GOgong neng zhu shi 、KEGGtong lu fu ji fen xi ,bing jie ge zhuai lu zu xue shu ju tan tao ARGzuo yong san dai chong qian zai zuo yong ba dian he fen zi ji zhi 。jie guo xian shi :（1）gong jian ding dao zong dan bai 2850ge ;yu kong bai dui zhao zu xiang bi ,4 mg/L,0.5 hchu li zu 、1.85 mg/L 0.5 hhe 4 hchu li zu fen bie yin qi 335ge 、223ge he 313ge cha yi biao da dan bai ,san ge ARGchu li zu zhong jun cha yi biao da dan bai 82ge 。（2）sheng wu xin xi xue fen xi jie guo xian shi cha yi biao da dan bai zhu yao can yu xi bao gu jia 、li zi zhuai yun yi ji ji rou shou su deng sheng li guo cheng 。（3）zhuai lu zu xue he dan bai zhi zu xue guan lian fen xi fa xian 19ge mRNAhe dan bai shui ping cha yi biao da yi zhi de dan bai ,ye shi ARGzhi jie huo jian jie de yao wu sha chong ba dian ,bei fu ji dao ji dong dan bai xi bao gu jia diao jie 、yang hua lin suan hua （oxidative phosphorylation）、nei tun zuo yong （Endocytosis）yi ji nian zhao ban （Focal adhesion）deng tong lu zhong 。ji zhong ATP synthase E chain（ATPge cheng mei Elian ）can yu ji ti ATPzhi jie ge cheng guo cheng 。（4）jin yi bu li yong qRT-PCRji shu yan jiu ARGdui san dai chong ATPge cheng mei bu tong ya ji yi ji hu xi lian fu ge wu Ⅰ-Ⅳji yin biao da de ying xiang ,jie guo fa xian ge ji yin jing 1.85 mg/Lde ARGchu li 6、12he 24 hhou jun xian zhe xia diao ;ci wai jing ARGli ti he zai ti chu li hou de san dai chong ti nei ATPhan liang jun ji ju jiang di ,biao ming ARGque shi neng yin qi san dai chong neng liang gong ying bu zu 。5.li yong jin na mi tan zhen jian ding niu bang zi gan yuan zuo yong xiao lin san dai chong ba dian li yong jin na mi tan zhen fa tong guo qin he ceng xi yuan li zhi jie cong san dai chong de zong dan bai zhong zhi jie fen li ARGzuo yong xiao lin san dai chong ba dian dan bai ,qu de de jie guo ru xia :（1）shou xian tong guo po huai ARGnei zhi huan huo de hua ge wu 2,ji jie gou yu ARGlei shi dan huo xing cha yi ju da (EC50wei 68.9 mg/L),shua wei yin xing dui zhao pai chu jia yang xing dan bai ;（2）ARGhe hua ge wu 2fen bie jin hang ce lian qiang ji xiu shi lian jie liu xin suan jie gou ,shi ji tong guo jin liu jian lian jie dao jin na mi ke li biao mian ,huo de jin na mi tan zhen GNP-6（ARGlian jie de jin na mi ke li ）he GNP-10（hua ge wu 2lian jie de jin na mi ke li ）;（3）gao fen bian tou she dian jing guan cha dao jin na mi tan zhen GNP-6cheng jun yun de qiu xing ,ping jun li jing wei 3.58 nm,ci wai ,hai ke guan cha dao GNP-6neng jin ru xiao lin san dai chong xi bao he deng bu wei ,ju reng bao chi sha mie xiao lin san dai chong huo xing ;（4）jiang GNP-6he GNP-10fen bie yu xiao lin san dai chong dan bai lie jie ye fu yo ,jing SDS-PAGEfen li ,huo de liang tiao GNP-6zhong te yi xing tiao dai ,tong guo ji zhi fu zhu ji guang jie xi dian li fei hang shi jian zhi pu （MALDI TOF/TOF MS）fen xi bing cai yong Mascot 2.2ruan jian sou suo Uniprotxiu li yin gan xian chong shu ju ku ,que ding le ARGde ba dian dan bai wei ji qiu dan bai （MyosinⅡ）he ji rou Mxian zhuang pei dan bai （Muscle M-line assembly protein,UNC-89）。zeng shang suo shu ,ben yan jiu ji yu gou jian de xiao lin san dai chong gan ran jin yu shi yan mo xing ,jin hang xing tai xue yan jiu 、zhuai lu zu xue he dan bai zhi xue zu guan lian fen xi yi ji ba dian dan bai de fen li jian ding ,chu bu que ding ARGsha mie yu lei chan shi xi chong zuo yong ba dian shi MyosinⅡhe UNC-89,jun yu wei chi ji qiu dan bai zheng chang jie gou you guan ,biao ming ARGsha chong ji zhi yu yao wu po huai ji qiu dan bai jie gou ,yin qi chong ti neng liang gong ying bu zu zui zhong si wang you guan 。shang shu jie guo wei ARGjin yi bu jie gou you hua di gong le chong yao can kao zi liao 。
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