本文主要研究内容
作者王梦雅（2019）在《亚急性瘤胃酸中毒对奶山羊肠道上皮屏障功能的影响及其转录组学研究》一文中研究指出：亚急性瘤胃酸中毒(Subacute rumen acidosis,SARA)是现代反刍动物集约化养殖中常见的一类营养代谢病,在国际上备受学者关注。前期该领域的研究工作仅限于SARA对反刍动物瘤胃上皮的影响,然而对其肠道上皮屏障功能影响的报道较为罕见。为此,本论文将从组织、分子以及组学层面系统研究SARA对奶山羊肠道上皮屏障功能的损伤及其可能调控机制。1.SARA对奶山羊肠道上皮形态结构的影响本试验选用12只体况良好、体重相近(62.13±4.76kg)、安装有永久性瘤胃瘘管的泌乳奶山羊,随机分成两组,即对照组(n=6)和SARA组(n=6)。对照组饲喂NFC/NDF为1.15的基础日粮,SARA组依次饲喂NFC/NDF为1.15、1.49、2.12和2.66的4种试验日粮诱导奶山羊发生SARA,每种日粮持续饲喂15 d,为期60 d,在每种日粮饲喂结束前3 d采用pH酸度计对瘤胃pH值进行连续监测,以此作为判定SARA发生的依据,当一天内瘤胃pH值在5.2～5.5持续时间长达3 h以上即视为SARA模型建立成功。在此基础上,分别选取对照组及SARA组各3只奶山羊禁食12 h后进行屠宰,采集十二指肠、空肠、盲肠和结肠各段中间部位上皮组织,利用光学显微镜和透射电镜技术,对肠道上皮形态结构、细胞间超微结构进行观察。结果表明:(1)SARA显著提高了奶山羊十二指肠和空肠的绒毛高度、隐窝深度(P<0.05),黏膜厚度在空肠中显著升高(P<0.05),在十二指肠中升高但无显著性差异(P>0.05);SARA降低了十二指肠(P>0.05)和空肠(P<0.05)的绒毛高度/隐窝深度比值,同时使盲肠和结肠上皮表面出现严重的细胞凹陷及隐窝坏死。(2)SARA使奶山羊十二指肠、空肠和结肠上皮细胞间紧密连接的电子密度降低,细胞间隙增大,线粒体肿胀,部分线粒体脊断裂或消失。以上结果表明:SARA破坏了肠道上皮的形态结构,使其完整性受损。2.SARA对奶山羊肠道上皮通透性的影响试验设计、试验动物及日粮均与试验一相同。利用Ussing chamber系统测定肠道上皮组织电生理参数和异硫氰酸荧光素-葡聚糖4(FD4)流速,并对血浆LPS、D-乳酸含量及DAO活性进行检测,以此来反映SARA对奶山羊肠道上皮通透性的影响。结果表明:(1)SARA组奶山羊十二指肠、空肠和结肠上皮的Isc、Gt和FD4流速显著高于对照组(P<0.05),其PD显著低于对照组(P<0.05)。(2)与对照组相比,SARA组奶山羊血浆LPS、D-乳酸含量及DAO活性均显著升高(P<0.05)。以上结果表明:发生SARA的奶山羊,其肠道上皮通透性显著增加。3.SARA对奶山羊肠道上皮细胞间紧密连接蛋白表达的影响试验设计、试验动物及日粮均与试验一相同。本试验分别采用实时荧光定量PCR(qRT-PCR)、免疫组化SP法及免疫印迹技术(Western-Blot)检测肠道上皮Claudin-1、Claudin-4、Claudin-7、Occludin 及 ZO-1 基因的 mRNA 表达水平和蛋白表达水平的变化,以期从分子水平揭示SARA对奶山羊肠道上皮屏障功能的损伤机制。结果显示:SARA显著上调了十二指肠上皮Claudin-1、Claudin-4、Claudin-7及ZO-1的mRNA表达水平和蛋白表达水平(P<0.05),上调了 Occludin的mRNA表达水平(P<0.05)和蛋白表达水平(P>0.05);同时显著上调了空肠上皮Claudin-4、Claudin-7、Occludin及ZO-1的mRNA表达水平和蛋白表达水平(P<0.05),下调了 Claudin-1的mRNA表达水平和蛋白表达水平,但无显著性差异(P>0.05);也上调了结肠上皮 Claudin-1(P<0.05)、Claudin-4(P>0.05)及 Occludin(P<0.05)的mRNA表达水平和蛋白表达水平,下调了Claudin-7(P>0.05)和ZO-1(P>0.05)的mRNA表达水平和蛋白表达水平。以上结果表明:SARA改变了肠道上皮紧密连接基因的mRNA表达水平和蛋白表达水平,且在不同肠段的表达水平有所不同。4.SARA对奶山羊十二指肠组织的转录组学分析本试验利用RNA-seq技术对奶山羊十二指肠组织进行转录组测序。结果表明:对照组和SARA组经过滤共获得30 GB的Clean reads。样本匹配到基因组的总基因数为26 724个,其中只在对照组表达的基因有273个,只在SARA组表达的基因有473个,在两个处理组中均表达的基因有12 989个。采用edgeR软件对基因表达量做差异表达分析,以显著水平P<0.05及|log2FC| ≥ 1为标准筛选差异表达基因(DEGs),结果共获得495个DEGs,其中有177个基因表达上调,318个基因表达下调。将筛选得到的DEGs进行GO功能注释和KEGG pathway富集分析,结果显示,这些上调基因主要与细胞凋亡、炎症反应、白细胞迁移等功能相关,而下调的基因主要与离子转运、脂肪酸代谢、脂质转运(代谢)、有机酸转运(代谢)等功能相关,并且这些DEGs主要富集到紧密连接信号通路、趋化因子信号通路和钙离子等信号通路上。上述结果表明:SARA破坏了肠道上皮的形态结构,导致肠道上皮屏障功能受损,通透性增加。
Abstract
ya ji xing liu wei suan zhong du (Subacute rumen acidosis,SARA)shi xian dai fan chu dong wu ji yao hua yang shi zhong chang jian de yi lei ying yang dai xie bing ,zai guo ji shang bei shou xue zhe guan zhu 。qian ji gai ling yu de yan jiu gong zuo jin xian yu SARAdui fan chu dong wu liu wei shang pi de ying xiang ,ran er dui ji chang dao shang pi bing zhang gong neng ying xiang de bao dao jiao wei han jian 。wei ci ,ben lun wen jiang cong zu zhi 、fen zi yi ji zu xue ceng mian ji tong yan jiu SARAdui nai shan yang chang dao shang pi bing zhang gong neng de sun shang ji ji ke neng diao kong ji zhi 。1.SARAdui nai shan yang chang dao shang pi xing tai jie gou de ying xiang ben shi yan shua yong 12zhi ti kuang liang hao 、ti chong xiang jin (62.13±4.76kg)、an zhuang you yong jiu xing liu wei lou guan de bi ru nai shan yang ,sui ji fen cheng liang zu ,ji dui zhao zu (n=6)he SARAzu (n=6)。dui zhao zu si wei NFC/NDFwei 1.15de ji chu ri liang ,SARAzu yi ci si wei NFC/NDFwei 1.15、1.49、2.12he 2.66de 4chong shi yan ri liang you dao nai shan yang fa sheng SARA,mei chong ri liang chi xu si wei 15 d,wei ji 60 d,zai mei chong ri liang si wei jie shu qian 3 dcai yong pHsuan du ji dui liu wei pHzhi jin hang lian xu jian ce ,yi ci zuo wei pan ding SARAfa sheng de yi ju ,dang yi tian nei liu wei pHzhi zai 5.2～5.5chi xu shi jian chang da 3 hyi shang ji shi wei SARAmo xing jian li cheng gong 。zai ci ji chu shang ,fen bie shua qu dui zhao zu ji SARAzu ge 3zhi nai shan yang jin shi 12 hhou jin hang tu zai ,cai ji shi er zhi chang 、kong chang 、mang chang he jie chang ge duan zhong jian bu wei shang pi zu zhi ,li yong guang xue xian wei jing he tou she dian jing ji shu ,dui chang dao shang pi xing tai jie gou 、xi bao jian chao wei jie gou jin hang guan cha 。jie guo biao ming :(1)SARAxian zhe di gao le nai shan yang shi er zhi chang he kong chang de rong mao gao du 、yin wo shen du (P<0.05),nian mo hou du zai kong chang zhong xian zhe sheng gao (P<0.05),zai shi er zhi chang zhong sheng gao dan mo xian zhe xing cha yi (P>0.05);SARAjiang di le shi er zhi chang (P>0.05)he kong chang (P<0.05)de rong mao gao du /yin wo shen du bi zhi ,tong shi shi mang chang he jie chang shang pi biao mian chu xian yan chong de xi bao ao xian ji yin wo huai si 。(2)SARAshi nai shan yang shi er zhi chang 、kong chang he jie chang shang pi xi bao jian jin mi lian jie de dian zi mi du jiang di ,xi bao jian xi zeng da ,xian li ti zhong zhang ,bu fen xian li ti ji duan lie huo xiao shi 。yi shang jie guo biao ming :SARApo huai le chang dao shang pi de xing tai jie gou ,shi ji wan zheng xing shou sun 。2.SARAdui nai shan yang chang dao shang pi tong tou xing de ying xiang shi yan she ji 、shi yan dong wu ji ri liang jun yu shi yan yi xiang tong 。li yong Ussing chamberji tong ce ding chang dao shang pi zu zhi dian sheng li can shu he yi liu qing suan ying guang su -pu ju tang 4(FD4)liu su ,bing dui xie jiang LPS、D-ru suan han liang ji DAOhuo xing jin hang jian ce ,yi ci lai fan ying SARAdui nai shan yang chang dao shang pi tong tou xing de ying xiang 。jie guo biao ming :(1)SARAzu nai shan yang shi er zhi chang 、kong chang he jie chang shang pi de Isc、Gthe FD4liu su xian zhe gao yu dui zhao zu (P<0.05),ji PDxian zhe di yu dui zhao zu (P<0.05)。(2)yu dui zhao zu xiang bi ,SARAzu nai shan yang xie jiang LPS、D-ru suan han liang ji DAOhuo xing jun xian zhe sheng gao (P<0.05)。yi shang jie guo biao ming :fa sheng SARAde nai shan yang ,ji chang dao shang pi tong tou xing xian zhe zeng jia 。3.SARAdui nai shan yang chang dao shang pi xi bao jian jin mi lian jie dan bai biao da de ying xiang shi yan she ji 、shi yan dong wu ji ri liang jun yu shi yan yi xiang tong 。ben shi yan fen bie cai yong shi shi ying guang ding liang PCR(qRT-PCR)、mian yi zu hua SPfa ji mian yi yin ji ji shu (Western-Blot)jian ce chang dao shang pi Claudin-1、Claudin-4、Claudin-7、Occludin ji ZO-1 ji yin de mRNA biao da shui ping he dan bai biao da shui ping de bian hua ,yi ji cong fen zi shui ping jie shi SARAdui nai shan yang chang dao shang pi bing zhang gong neng de sun shang ji zhi 。jie guo xian shi :SARAxian zhe shang diao le shi er zhi chang shang pi Claudin-1、Claudin-4、Claudin-7ji ZO-1de mRNAbiao da shui ping he dan bai biao da shui ping (P<0.05),shang diao le Occludinde mRNAbiao da shui ping (P<0.05)he dan bai biao da shui ping (P>0.05);tong shi xian zhe shang diao le kong chang shang pi Claudin-4、Claudin-7、Occludinji ZO-1de mRNAbiao da shui ping he dan bai biao da shui ping (P<0.05),xia diao le Claudin-1de mRNAbiao da shui ping he dan bai biao da shui ping ,dan mo xian zhe xing cha yi (P>0.05);ye shang diao le jie chang shang pi Claudin-1(P<0.05)、Claudin-4(P>0.05)ji Occludin(P<0.05)de mRNAbiao da shui ping he dan bai biao da shui ping ,xia diao le Claudin-7(P>0.05)he ZO-1(P>0.05)de mRNAbiao da shui ping he dan bai biao da shui ping 。yi shang jie guo biao ming :SARAgai bian le chang dao shang pi jin mi lian jie ji yin de mRNAbiao da shui ping he dan bai biao da shui ping ,ju zai bu tong chang duan de biao da shui ping you suo bu tong 。4.SARAdui nai shan yang shi er zhi chang zu zhi de zhuai lu zu xue fen xi ben shi yan li yong RNA-seqji shu dui nai shan yang shi er zhi chang zu zhi jin hang zhuai lu zu ce xu 。jie guo biao ming :dui zhao zu he SARAzu jing guo lv gong huo de 30 GBde Clean reads。yang ben pi pei dao ji yin zu de zong ji yin shu wei 26 724ge ,ji zhong zhi zai dui zhao zu biao da de ji yin you 273ge ,zhi zai SARAzu biao da de ji yin you 473ge ,zai liang ge chu li zu zhong jun biao da de ji yin you 12 989ge 。cai yong edgeRruan jian dui ji yin biao da liang zuo cha yi biao da fen xi ,yi xian zhe shui ping P<0.05ji |log2FC| ≥ 1wei biao zhun shai shua cha yi biao da ji yin (DEGs),jie guo gong huo de 495ge DEGs,ji zhong you 177ge ji yin biao da shang diao ,318ge ji yin biao da xia diao 。jiang shai shua de dao de DEGsjin hang GOgong neng zhu shi he KEGG pathwayfu ji fen xi ,jie guo xian shi ,zhe xie shang diao ji yin zhu yao yu xi bao diao wang 、yan zheng fan ying 、bai xi bao qian yi deng gong neng xiang guan ,er xia diao de ji yin zhu yao yu li zi zhuai yun 、zhi fang suan dai xie 、zhi zhi zhuai yun (dai xie )、you ji suan zhuai yun (dai xie )deng gong neng xiang guan ,bing ju zhe xie DEGszhu yao fu ji dao jin mi lian jie xin hao tong lu 、qu hua yin zi xin hao tong lu he gai li zi deng xin hao tong lu shang 。shang shu jie guo biao ming :SARApo huai le chang dao shang pi de xing tai jie gou ,dao zhi chang dao shang pi bing zhang gong neng shou sun ,tong tou xing zeng jia 。
论文参考文献
论文详细介绍
论文作者分别是来自内蒙古农业大学的王梦雅，发表于刊物内蒙古农业大学2019-10-08论文，是一篇关于亚急性瘤胃酸中毒论文,肠道上皮论文,尤斯灌流系统论文,屏障功能论文,通透性论文,转录组测序论文,奶山羊论文,内蒙古农业大学2019-10-08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，资料来自内蒙古农业大学2019-10-08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：亚急性瘤胃酸中毒论文; 肠道上皮论文; 尤斯灌流系统论文; 屏障功能论文; 通透性论文; 转录组测序论文; 奶山羊论文; 内蒙古农业大学2019-10-08论文;