本文主要研究内容
作者高强（2019）在《猪种布鲁氏菌感染相关循环miRNA的筛选及验证》一文中研究指出：布鲁氏菌病(Brucellosis)是一种由布鲁氏菌(Brucella)引起的烈性人畜共患传染病,广泛流行于全球160多个国家和地区,给世界范围内畜牧业的发展和人类的健康带来了巨大的威胁。目前,布鲁氏菌病的诊断方法主要可以分为病原学诊断和血清学诊断,但两类方法各自存在着一些缺点与不足:病原学诊断存在病原分离操作难度大,分离成功率低,病原培养时间长以及存在一定生物安全风险等问题;血清学诊断则存在凝集抗原容易和多种与布鲁氏菌存在相似抗原表位的细菌抗血清(大肠杆菌O157、耶尔森菌O9等)发生交叉反应、不能区分免疫抗体和感染抗体等问题。microRNA(简称miRNA)是一类长约22nt的内源性非编码小分子RNA,广泛存在于线虫、果蝇、植物和包括人在内的真核生物中。随着对其研究的深入,人们发现miRNA可以从细胞内释放,广泛而稳定的存在于包括血清、血浆、唾液、尿液甚至牛奶等细胞外液中,人们将这类miRNA统称为循环miRNA(circulating microRNA)。循环miRNA具有存在稳定、物种间进化保守、检测方法多样等特点,并且循环miRNA作为一种新的疾病诊断标志物已经在人类医学很多领域有了相关研究。因此,基于目前两类布鲁氏菌病诊断方法存在的缺陷以及循环miRNA在疾病领域研究的现状,我们希望探究布鲁氏菌感染宿主后,宿主体内某些循环miRNA表达量的相对变化,从而探索将此类循环miRNA作为布鲁氏菌感染诊断标志物的可能。本研究首先建立了布鲁氏菌感染BALB/c小鼠的模型,分别用猪种布鲁氏菌S2株和S1330株感染BALB/c小鼠,同时设立同等数量的空白对照组。在感染后的第7d、14d、21d、28d,分别取三组小鼠每组各5只,每只小鼠进行眼球采血分离血清、无菌摘除脾脏称取脾脏重量、无菌研磨脾脏计算脾脏含菌量操作,分别得到三组小鼠在四个时间点血清抗体水平、脾脏重量以及脾脏含菌量的变化趋势。根据这三组数据反映的趋势,确定将第28d的血清样品送样进行转录组学测序,血清转录组测序结果显示:S1330株感染组和空白对照组小鼠血清中有15个miRNA表达量发生显著变化,其中9个上调,6个下调;S2株感染组和空白对照组小鼠血清中有11个miRNA表达量发生显著变化,其中4个上调,7个下调;S1330株感染组和S2株感染组小鼠血清中有3个miRNA表达量发生显著变化,其中1个上调,2个下调。根据转录组学测序结果,我们在三组对照中分别挑选了10条,2条,2条表达量发生显著变化的miRNA进行验证。在miRBase数据库中查询相关序列信息并根据序列设计出检测这些miRNA的引物,通过小鼠血清miRNA提取,反转录,荧光定量PCR以及扩大样本检测量等实验,最终在S1330株感染组和空白对照组中筛选出了一条与转录组测序结果匹配并且表达量存在显著差异(上调)的miRNA,即mmu-miR-10b-5p。同时,我们也对mmu-miR-10b-5p在小鼠巨噬细胞RAW264.7感染猪种布鲁氏菌S1330株前后的表达水平进行了初探,结果显示,巨噬细胞在感染S1330株48h后,感染组和空白对照组中mmu-miR-10b-5p的表达水平也出现了显著差异。之后,我们对巨噬细胞内mmu-miR-10b-5p的下游靶点基因进行了预测,在预测出的52个下游靶点基因中,验证出了6个基因的相对表达量出现了显著下调,这6个基因的功能大多与细胞凋亡和炎性反应相关。最后,我们对临床布病阳性和阴性牛血清样本中的bta-miR-10b-5p的表达水平进行了检测,但是其表达水平并没有表现出显著差异。综上,通过本论文研究,成功地在S1330株感染组和空白对照组小鼠血清中筛选出了一条表达量存在显著差异(上调)的miRNA,为将循环miRNA作为一类布鲁氏菌感染诊断标志物的研究提供了思路。
Abstract
bu lu shi jun bing (Brucellosis)shi yi chong you bu lu shi jun (Brucella)yin qi de lie xing ren chu gong huan chuan ran bing ,an fan liu hang yu quan qiu 160duo ge guo jia he de ou ,gei shi jie fan wei nei chu mu ye de fa zhan he ren lei de jian kang dai lai le ju da de wei xie 。mu qian ,bu lu shi jun bing de zhen duan fang fa zhu yao ke yi fen wei bing yuan xue zhen duan he xie qing xue zhen duan ,dan liang lei fang fa ge zi cun zai zhao yi xie que dian yu bu zu :bing yuan xue zhen duan cun zai bing yuan fen li cao zuo nan du da ,fen li cheng gong lv di ,bing yuan pei yang shi jian chang yi ji cun zai yi ding sheng wu an quan feng xian deng wen ti ;xie qing xue zhen duan ze cun zai ning ji kang yuan rong yi he duo chong yu bu lu shi jun cun zai xiang shi kang yuan biao wei de xi jun kang xie qing (da chang gan jun O157、ye er sen jun O9deng )fa sheng jiao cha fan ying 、bu neng ou fen mian yi kang ti he gan ran kang ti deng wen ti 。microRNA(jian chen miRNA)shi yi lei chang yao 22ntde nei yuan xing fei bian ma xiao fen zi RNA,an fan cun zai yu xian chong 、guo ying 、zhi wu he bao gua ren zai nei de zhen he sheng wu zhong 。sui zhao dui ji yan jiu de shen ru ,ren men fa xian miRNAke yi cong xi bao nei shi fang ,an fan er wen ding de cun zai yu bao gua xie qing 、xie jiang 、tuo ye 、niao ye shen zhi niu nai deng xi bao wai ye zhong ,ren men jiang zhe lei miRNAtong chen wei xun huan miRNA(circulating microRNA)。xun huan miRNAju you cun zai wen ding 、wu chong jian jin hua bao shou 、jian ce fang fa duo yang deng te dian ,bing ju xun huan miRNAzuo wei yi chong xin de ji bing zhen duan biao zhi wu yi jing zai ren lei yi xue hen duo ling yu you le xiang guan yan jiu 。yin ci ,ji yu mu qian liang lei bu lu shi jun bing zhen duan fang fa cun zai de que xian yi ji xun huan miRNAzai ji bing ling yu yan jiu de xian zhuang ,wo men xi wang tan jiu bu lu shi jun gan ran su zhu hou ,su zhu ti nei mou xie xun huan miRNAbiao da liang de xiang dui bian hua ,cong er tan suo jiang ci lei xun huan miRNAzuo wei bu lu shi jun gan ran zhen duan biao zhi wu de ke neng 。ben yan jiu shou xian jian li le bu lu shi jun gan ran BALB/cxiao shu de mo xing ,fen bie yong zhu chong bu lu shi jun S2zhu he S1330zhu gan ran BALB/cxiao shu ,tong shi she li tong deng shu liang de kong bai dui zhao zu 。zai gan ran hou de di 7d、14d、21d、28d,fen bie qu san zu xiao shu mei zu ge 5zhi ,mei zhi xiao shu jin hang yan qiu cai xie fen li xie qing 、mo jun zhai chu pi zang chen qu pi zang chong liang 、mo jun yan mo pi zang ji suan pi zang han jun liang cao zuo ,fen bie de dao san zu xiao shu zai si ge shi jian dian xie qing kang ti shui ping 、pi zang chong liang yi ji pi zang han jun liang de bian hua qu shi 。gen ju zhe san zu shu ju fan ying de qu shi ,que ding jiang di 28dde xie qing yang pin song yang jin hang zhuai lu zu xue ce xu ,xie qing zhuai lu zu ce xu jie guo xian shi :S1330zhu gan ran zu he kong bai dui zhao zu xiao shu xie qing zhong you 15ge miRNAbiao da liang fa sheng xian zhe bian hua ,ji zhong 9ge shang diao ,6ge xia diao ;S2zhu gan ran zu he kong bai dui zhao zu xiao shu xie qing zhong you 11ge miRNAbiao da liang fa sheng xian zhe bian hua ,ji zhong 4ge shang diao ,7ge xia diao ;S1330zhu gan ran zu he S2zhu gan ran zu xiao shu xie qing zhong you 3ge miRNAbiao da liang fa sheng xian zhe bian hua ,ji zhong 1ge shang diao ,2ge xia diao 。gen ju zhuai lu zu xue ce xu jie guo ,wo men zai san zu dui zhao zhong fen bie tiao shua le 10tiao ,2tiao ,2tiao biao da liang fa sheng xian zhe bian hua de miRNAjin hang yan zheng 。zai miRBaseshu ju ku zhong cha xun xiang guan xu lie xin xi bing gen ju xu lie she ji chu jian ce zhe xie miRNAde yin wu ,tong guo xiao shu xie qing miRNAdi qu ,fan zhuai lu ,ying guang ding liang PCRyi ji kuo da yang ben jian ce liang deng shi yan ,zui zhong zai S1330zhu gan ran zu he kong bai dui zhao zu zhong shai shua chu le yi tiao yu zhuai lu zu ce xu jie guo pi pei bing ju biao da liang cun zai xian zhe cha yi (shang diao )de miRNA,ji mmu-miR-10b-5p。tong shi ,wo men ye dui mmu-miR-10b-5pzai xiao shu ju shi xi bao RAW264.7gan ran zhu chong bu lu shi jun S1330zhu qian hou de biao da shui ping jin hang le chu tan ,jie guo xian shi ,ju shi xi bao zai gan ran S1330zhu 48hhou ,gan ran zu he kong bai dui zhao zu zhong mmu-miR-10b-5pde biao da shui ping ye chu xian le xian zhe cha yi 。zhi hou ,wo men dui ju shi xi bao nei mmu-miR-10b-5pde xia you ba dian ji yin jin hang le yu ce ,zai yu ce chu de 52ge xia you ba dian ji yin zhong ,yan zheng chu le 6ge ji yin de xiang dui biao da liang chu xian le xian zhe xia diao ,zhe 6ge ji yin de gong neng da duo yu xi bao diao wang he yan xing fan ying xiang guan 。zui hou ,wo men dui lin chuang bu bing yang xing he yin xing niu xie qing yang ben zhong de bta-miR-10b-5pde biao da shui ping jin hang le jian ce ,dan shi ji biao da shui ping bing mei you biao xian chu xian zhe cha yi 。zeng shang ,tong guo ben lun wen yan jiu ,cheng gong de zai S1330zhu gan ran zu he kong bai dui zhao zu xiao shu xie qing zhong shai shua chu le yi tiao biao da liang cun zai xian zhe cha yi (shang diao )de miRNA,wei jiang xun huan miRNAzuo wei yi lei bu lu shi jun gan ran zhen duan biao zhi wu de yan jiu di gong le sai lu 。
论文参考文献
论文详细介绍
论文作者分别是来自中国兽医药品监察所的高强，发表于刊物中国兽医药品监察所2019-08-01论文，是一篇关于布鲁氏菌病论文,循环论文,诊断标志物论文,转录组测序论文,中国兽医药品监察所2019-08-01论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自中国兽医药品监察所2019-08-01论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：布鲁氏菌病论文; 循环论文; 诊断标志物论文; 转录组测序论文; 中国兽医药品监察所2019-08-01论文;