本文主要研究内容
作者王加众（2019）在《Diutina rugosa SD-17的生物学特性及其益生作用的研究》一文中研究指出：益生菌被定义为当以适当的量施用时,赋予宿主健康益处的活的微生物。通常指的益生菌为益生性细菌,由于细菌培养方式简单、增殖速度快、环境耐受性强等特点,被广泛开发和应用。但是益生性细菌也有其致命的弱点,例如基因转移,容易导致抗性基因的传播。真菌具有遗传隔离的特性,能够有效避免抗性基因的转移。真菌细胞壁中的β-葡聚糖已被证明是一种高效的益生物质,能够调节机体的免疫水平。另外,真菌作为载体表达的基因工程产物具有天然构象,可以作为良好的疫苗输送平台。这些都表明真菌具有作为优良益生菌被开发的潜力。益生菌的作用机制是复杂的,但主要包括以下几个方面。第一,增重和提高饲料利用率是开发畜禽肠道益生菌的首要目的,所以提高生长性能是权衡益生效果的重要指标。第二,消化系统的发育状况影响机体对营养物质的吸收和利用,促进消化系统的发育,也是评价益生菌的一个因素。第三,免疫状态直接决定机体的健康水平,益生菌株应该能够调节宿主的免疫水平。最后,肠道菌群影响机体代谢和对营养物质的吸收,益生菌株能够维持肠道菌群的平衡,并促进肠道菌群向有益的方向发展。现阶段,益生菌株主要从自然界分离获得,通过随机诱变或者基因定向改造获取益生菌株的方式比较少见。本实验室前期从雏鸡粪便中分离得到一株D.rugosa SD,预实验发现D.rugosa SD在雏鸡肠道不定植且不耐受肠道恶劣内环境。本课题基于此菌株,通过诱变筛选获得肠道定植能力强的变异株,旨在进一步研究其菌株特异性及其益生作用机制。1.肠道定植菌株筛选及菌株特异性分析本研究以存活率50%~70%为标准,确定了UV及NTG诱变剂的最佳诱变条件:15J,0.4min和50 mg/m L,40 min。进而以复合诱变的方式,以在雏鸡肠道内定植12 h为筛选条件,成功筛选到23株突变菌株。经过20次遗传稳定性检测,最终获得一株能够稳定遗传的变异菌株。通过物种进化分析发现,变异菌株与模式菌株D.rugosa ATCC10571的亲缘关系最近(自展值100%),最终命名为D.rugosa SD-17。D.rugosa SD-17生物学特性发生了显著变化,主要表现在:D.rugosa SD-17在41℃的增殖能力显著增强;D.rugosa SD-17在YTD固体培养基上的菌落中央出现明显的凹陷;经过透射电子显微镜发现D.rugosa SD-17的细胞壁显著增厚;D.rugosa SD-17的细胞壁及其β-葡聚糖含量显著提高。2.D.rugosa SD-17的益生潜力研究为了进一步探究D.rugosa SD-17的益生潜力,本研究通过模拟肠道内环境(胃酸、胆盐和温度)的方法来研究D.rugosa SD-17的耐受性。结果表明经过人工胃酸(p H=2)处理8 h,D.rugosa SD-17的存活率仍接近50%;0.3%的人工胆盐对D.rugosa SD-17的增殖没有显著影响;D.rugosa SD-17在畜禽肠道温度(37℃~41℃)下能够良好生长。通过细胞粘附试验发现,D.rugosa SD-17对Caco-2细胞的粘附率为61.5%,显著高于模式菌株。使用D.rugosa SD-17灌胃雏鸡,并在16 h后收集肠内容物涂板培养,结果从回肠内容物中分离出大量的D.rugosa SD-17,这说明D.rugosa SD-17在雏鸡肠道内的主要定植部位为回肠。以上结果表明D.rugosa SD-17对模拟的肠道内环境有良好的耐受力并能够在雏鸡肠道内短暂定植。3.D.rugosa SD-17对雏鸡的益生作用研究本研究通过急性毒性试验和慢性感染性试验来评价D.rugosa SD-17对雏鸡的安全性。结果均未引起雏鸡死亡,另外,雏鸡精神状态良好、采食量正常、表观和内脏无病理性变化、全血检测指标正常,这些结果说明D.rugosa SD-17对雏鸡没有致病性。动物益生性试验发现,饲料中添加D.rugosa SD-17会显著提高雏鸡增重和饲料利用率,并且促进回肠绒毛的发育,回肠恰巧也是D.rugosa SD-17最主要的定植部位,这表明该菌株对回肠上皮细胞可能有一定的刺激作用。在刀豆蛋白A(Con A)的刺激下,D.rugosa SD-17组的淋巴细胞增殖能力显著提高。通过流式细胞术检测淋巴细胞亚群,结果发现D.rugosa SD-17会显著提高CD3+CD4+细胞(T helper cells,Th)的比例。另外,通过荧光定量PCR检测Th细胞表面的标志性细胞因子,结果发现D.rugosa SD-17会促进Th1细胞标志性细胞因子的基因表达量,而对Th2细胞标志性细胞因子的基因表达量没有影响,这些结果表明D.rugosa SD-17对雏鸡有一定的免疫调节作用。4.D.rugosa SD-17对肠道菌群的影响本研究通过16S扩增子测序分析回肠菌群、盲肠菌群、结肠菌群和粪便菌群的变化来揭示D.rugosa SD-17对小鼠的肠道内环境的影响。首先,D.rugosa SD-17的刺激引起回肠菌群丰富度的显著提高。其次,D.rugosa SD-17的刺激会引起粪便菌群中厚壁菌门的比例升高,拟杆菌门的比例降低,这有利于提高机体对能量的吸收和利用。另外,D.rugosa SD-17的刺激会引起有益菌属(乳酸杆菌属)比例的显著升高。这些结果初步表明,D.rugosa SD-17能够调整小鼠肠道的菌群结构,并增加有益菌群的比例。5.D.rugosa SD-17对代谢通路的影响根据小鼠肠道菌群测序的结果,本研究通过KEEG数据库进行富集分析,旨在揭示D.rugosa SD-17对小鼠代谢水平的影响。从结肠菌群中富集到5个D.rugosa SD-17特异性代谢通路,分别涉及细胞粘附分子、细胞骨架的调节、病毒的感染及心肌病等代谢领域。本研究选取细胞粘附分子通路进行分析,发现E-cadherin是肠道粘附连接的核心蛋白。为了进一步研究D.rugosa SD-17对小鼠结肠粘附连接的影响,我们用D.rugosa SD-17分别刺激结肠组织和Caco-2细胞,结果都显示E-cadherin表达量显著提高。另外,粘附连接的其它相关蛋白(β-catenin、ZO-1等)的表达量都发生了不同程度的显著上调。这些结果表明D.rugosa SD-17显著提高结肠粘附连接的相关蛋白的基因表达量,对维持肠道结构有一定的促进作用。综上所述,本课题成功获取一株肠道定植能力强的益生性菌株D.rugosa SD-17,该菌株具有明显的菌株特异性,对雏鸡有良好的益生作用,并能够优化肠道菌群和维持肠道结构。本研究为深入研究畜禽肠道益生菌株的作用机制奠定基础,并为畜禽肠道益生菌的开发和应用提供指导意义。
Abstract
yi sheng jun bei ding yi wei dang yi kuo dang de liang shi yong shi ,fu yu su zhu jian kang yi chu de huo de wei sheng wu 。tong chang zhi de yi sheng jun wei yi sheng xing xi jun ,you yu xi jun pei yang fang shi jian chan 、zeng shi su du kuai 、huan jing nai shou xing jiang deng te dian ,bei an fan kai fa he ying yong 。dan shi yi sheng xing xi jun ye you ji zhi ming de ruo dian ,li ru ji yin zhuai yi ,rong yi dao zhi kang xing ji yin de chuan bo 。zhen jun ju you wei chuan ge li de te xing ,neng gou you xiao bi mian kang xing ji yin de zhuai yi 。zhen jun xi bao bi zhong de β-pu ju tang yi bei zheng ming shi yi chong gao xiao de yi sheng wu zhi ,neng gou diao jie ji ti de mian yi shui ping 。ling wai ,zhen jun zuo wei zai ti biao da de ji yin gong cheng chan wu ju you tian ran gou xiang ,ke yi zuo wei liang hao de yi miao shu song ping tai 。zhe xie dou biao ming zhen jun ju you zuo wei you liang yi sheng jun bei kai fa de qian li 。yi sheng jun de zuo yong ji zhi shi fu za de ,dan zhu yao bao gua yi xia ji ge fang mian 。di yi ,zeng chong he di gao si liao li yong lv shi kai fa chu qin chang dao yi sheng jun de shou yao mu de ,suo yi di gao sheng chang xing neng shi quan heng yi sheng xiao guo de chong yao zhi biao 。di er ,xiao hua ji tong de fa yo zhuang kuang ying xiang ji ti dui ying yang wu zhi de xi shou he li yong ,cu jin xiao hua ji tong de fa yo ,ye shi ping jia yi sheng jun de yi ge yin su 。di san ,mian yi zhuang tai zhi jie jue ding ji ti de jian kang shui ping ,yi sheng jun zhu ying gai neng gou diao jie su zhu de mian yi shui ping 。zui hou ,chang dao jun qun ying xiang ji ti dai xie he dui ying yang wu zhi de xi shou ,yi sheng jun zhu neng gou wei chi chang dao jun qun de ping heng ,bing cu jin chang dao jun qun xiang you yi de fang xiang fa zhan 。xian jie duan ,yi sheng jun zhu zhu yao cong zi ran jie fen li huo de ,tong guo sui ji you bian huo zhe ji yin ding xiang gai zao huo qu yi sheng jun zhu de fang shi bi jiao shao jian 。ben shi yan shi qian ji cong chu ji fen bian zhong fen li de dao yi zhu D.rugosa SD,yu shi yan fa xian D.rugosa SDzai chu ji chang dao bu ding zhi ju bu nai shou chang dao e lie nei huan jing 。ben ke ti ji yu ci jun zhu ,tong guo you bian shai shua huo de chang dao ding zhi neng li jiang de bian yi zhu ,zhi zai jin yi bu yan jiu ji jun zhu te yi xing ji ji yi sheng zuo yong ji zhi 。1.chang dao ding zhi jun zhu shai shua ji jun zhu te yi xing fen xi ben yan jiu yi cun huo lv 50%~70%wei biao zhun ,que ding le UVji NTGyou bian ji de zui jia you bian tiao jian :15J,0.4minhe 50 mg/m L,40 min。jin er yi fu ge you bian de fang shi ,yi zai chu ji chang dao nei ding zhi 12 hwei shai shua tiao jian ,cheng gong shai shua dao 23zhu tu bian jun zhu 。jing guo 20ci wei chuan wen ding xing jian ce ,zui zhong huo de yi zhu neng gou wen ding wei chuan de bian yi jun zhu 。tong guo wu chong jin hua fen xi fa xian ,bian yi jun zhu yu mo shi jun zhu D.rugosa ATCC10571de qin yuan guan ji zui jin (zi zhan zhi 100%),zui zhong ming ming wei D.rugosa SD-17。D.rugosa SD-17sheng wu xue te xing fa sheng le xian zhe bian hua ,zhu yao biao xian zai :D.rugosa SD-17zai 41℃de zeng shi neng li xian zhe zeng jiang ;D.rugosa SD-17zai YTDgu ti pei yang ji shang de jun la zhong yang chu xian ming xian de ao xian ;jing guo tou she dian zi xian wei jing fa xian D.rugosa SD-17de xi bao bi xian zhe zeng hou ;D.rugosa SD-17de xi bao bi ji ji β-pu ju tang han liang xian zhe di gao 。2.D.rugosa SD-17de yi sheng qian li yan jiu wei le jin yi bu tan jiu D.rugosa SD-17de yi sheng qian li ,ben yan jiu tong guo mo ni chang dao nei huan jing (wei suan 、dan yan he wen du )de fang fa lai yan jiu D.rugosa SD-17de nai shou xing 。jie guo biao ming jing guo ren gong wei suan (p H=2)chu li 8 h,D.rugosa SD-17de cun huo lv reng jie jin 50%;0.3%de ren gong dan yan dui D.rugosa SD-17de zeng shi mei you xian zhe ying xiang ;D.rugosa SD-17zai chu qin chang dao wen du (37℃~41℃)xia neng gou liang hao sheng chang 。tong guo xi bao nian fu shi yan fa xian ,D.rugosa SD-17dui Caco-2xi bao de nian fu lv wei 61.5%,xian zhe gao yu mo shi jun zhu 。shi yong D.rugosa SD-17guan wei chu ji ,bing zai 16 hhou shou ji chang nei rong wu tu ban pei yang ,jie guo cong hui chang nei rong wu zhong fen li chu da liang de D.rugosa SD-17,zhe shui ming D.rugosa SD-17zai chu ji chang dao nei de zhu yao ding zhi bu wei wei hui chang 。yi shang jie guo biao ming D.rugosa SD-17dui mo ni de chang dao nei huan jing you liang hao de nai shou li bing neng gou zai chu ji chang dao nei duan zan ding zhi 。3.D.rugosa SD-17dui chu ji de yi sheng zuo yong yan jiu ben yan jiu tong guo ji xing du xing shi yan he man xing gan ran xing shi yan lai ping jia D.rugosa SD-17dui chu ji de an quan xing 。jie guo jun wei yin qi chu ji si wang ,ling wai ,chu ji jing shen zhuang tai liang hao 、cai shi liang zheng chang 、biao guan he nei zang mo bing li xing bian hua 、quan xie jian ce zhi biao zheng chang ,zhe xie jie guo shui ming D.rugosa SD-17dui chu ji mei you zhi bing xing 。dong wu yi sheng xing shi yan fa xian ,si liao zhong tian jia D.rugosa SD-17hui xian zhe di gao chu ji zeng chong he si liao li yong lv ,bing ju cu jin hui chang rong mao de fa yo ,hui chang qia qiao ye shi D.rugosa SD-17zui zhu yao de ding zhi bu wei ,zhe biao ming gai jun zhu dui hui chang shang pi xi bao ke neng you yi ding de ci ji zuo yong 。zai dao dou dan bai A(Con A)de ci ji xia ,D.rugosa SD-17zu de lin ba xi bao zeng shi neng li xian zhe di gao 。tong guo liu shi xi bao shu jian ce lin ba xi bao ya qun ,jie guo fa xian D.rugosa SD-17hui xian zhe di gao CD3+CD4+xi bao (T helper cells,Th)de bi li 。ling wai ,tong guo ying guang ding liang PCRjian ce Thxi bao biao mian de biao zhi xing xi bao yin zi ,jie guo fa xian D.rugosa SD-17hui cu jin Th1xi bao biao zhi xing xi bao yin zi de ji yin biao da liang ,er dui Th2xi bao biao zhi xing xi bao yin zi de ji yin biao da liang mei you ying xiang ,zhe xie jie guo biao ming D.rugosa SD-17dui chu ji you yi ding de mian yi diao jie zuo yong 。4.D.rugosa SD-17dui chang dao jun qun de ying xiang ben yan jiu tong guo 16Skuo zeng zi ce xu fen xi hui chang jun qun 、mang chang jun qun 、jie chang jun qun he fen bian jun qun de bian hua lai jie shi D.rugosa SD-17dui xiao shu de chang dao nei huan jing de ying xiang 。shou xian ,D.rugosa SD-17de ci ji yin qi hui chang jun qun feng fu du de xian zhe di gao 。ji ci ,D.rugosa SD-17de ci ji hui yin qi fen bian jun qun zhong hou bi jun men de bi li sheng gao ,ni gan jun men de bi li jiang di ,zhe you li yu di gao ji ti dui neng liang de xi shou he li yong 。ling wai ,D.rugosa SD-17de ci ji hui yin qi you yi jun shu (ru suan gan jun shu )bi li de xian zhe sheng gao 。zhe xie jie guo chu bu biao ming ,D.rugosa SD-17neng gou diao zheng xiao shu chang dao de jun qun jie gou ,bing zeng jia you yi jun qun de bi li 。5.D.rugosa SD-17dui dai xie tong lu de ying xiang gen ju xiao shu chang dao jun qun ce xu de jie guo ,ben yan jiu tong guo KEEGshu ju ku jin hang fu ji fen xi ,zhi zai jie shi D.rugosa SD-17dui xiao shu dai xie shui ping de ying xiang 。cong jie chang jun qun zhong fu ji dao 5ge D.rugosa SD-17te yi xing dai xie tong lu ,fen bie she ji xi bao nian fu fen zi 、xi bao gu jia de diao jie 、bing du de gan ran ji xin ji bing deng dai xie ling yu 。ben yan jiu shua qu xi bao nian fu fen zi tong lu jin hang fen xi ,fa xian E-cadherinshi chang dao nian fu lian jie de he xin dan bai 。wei le jin yi bu yan jiu D.rugosa SD-17dui xiao shu jie chang nian fu lian jie de ying xiang ,wo men yong D.rugosa SD-17fen bie ci ji jie chang zu zhi he Caco-2xi bao ,jie guo dou xian shi E-cadherinbiao da liang xian zhe di gao 。ling wai ,nian fu lian jie de ji ta xiang guan dan bai (β-catenin、ZO-1deng )de biao da liang dou fa sheng le bu tong cheng du de xian zhe shang diao 。zhe xie jie guo biao ming D.rugosa SD-17xian zhe di gao jie chang nian fu lian jie de xiang guan dan bai de ji yin biao da liang ,dui wei chi chang dao jie gou you yi ding de cu jin zuo yong 。zeng shang suo shu ,ben ke ti cheng gong huo qu yi zhu chang dao ding zhi neng li jiang de yi sheng xing jun zhu D.rugosa SD-17,gai jun zhu ju you ming xian de jun zhu te yi xing ,dui chu ji you liang hao de yi sheng zuo yong ,bing neng gou you hua chang dao jun qun he wei chi chang dao jie gou 。ben yan jiu wei shen ru yan jiu chu qin chang dao yi sheng jun zhu de zuo yong ji zhi dian ding ji chu ,bing wei chu qin chang dao yi sheng jun de kai fa he ying yong di gong zhi dao yi yi 。
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论文作者分别是来自山东农业大学的王加众，发表于刊物山东农业大学2019-07-08论文，是一篇关于筛选论文,环境耐受性论文,益生作用论文,肠道菌群论文,钙粘蛋白论文,山东农业大学2019-07-08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，资料来自山东农业大学2019-07-08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。
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