本文主要研究内容
作者卢红（2019）在《APS缓解LPS诱导的BEAS-2B细胞内质网应激炎症反应机制》一文中研究指出：集约化养殖密度的增加加重了家禽呼吸系统疾病压力。家禽肺损伤是严重的呼吸系统病症,通常由环境变化、应激、病原感染等多种因素引起。其发病机制受多细胞器、细胞因子和信号通路调控。内质网应激信号通路是介导气道结构细胞命运的重要途径,区别于其它两条调控细胞命运的线粒体途径和死亡受体途径。表观遗传学参与多种生命活动(细胞生长、分化、凋亡),旨在从分子水平解释临床症状。本研究中,我们利用脂多糖(lipopolysaccharide,LPS)诱导人正常肺上皮细胞(BEAS-2B)构建体外内质网应激(endoplasmic retieulumstress,ERS)模型,分析表观遗传修饰酶的表达变化及其生物学功能,并探讨黄芪多糖(astragalus polysaccharides,APS)对LPS刺激的促炎性细胞因子的调控作用,以期为营养表观遗传调控肺部炎症反应奠定理论基础。试验一LPS诱导BEAS-2B细胞内质网应激模型的构建本试验首先检测LPS刺激BEAS-2B细胞ERS发生标志性蛋白免疫球蛋白重链结合蛋白(immunoglobulin heavy chain binding protein,BIP)的表达情况,构建ERS模型;其次,分析未折叠蛋白反应(unfolded protein response,UPR)信号通路蛋白肌醇需要酶1α(inositol-requiring enzyme 1α,IRE1α)、蛋白激酶RNA样内质网激酶(the kinase PKR-like ER-resident kinase,PERK)和激活转录因子6(activating transcription factor 6,ATF6)的表达情况,探讨LPS对BEAS-2B细胞ERS的影响及作用机制。蛋白水平研究LPS添加对BEAS-2B细胞ERS相关蛋白表达的剂量-效应关系和时间-效应关系。剂量-效应关系的结果显示,100 ng/m L LPS、200 ng/m L LPS和500 ng/m L LPS以剂量依赖性方式增加BEAS-2B细胞中BIP蛋白的表达(P<0.01)。添加200ng/m L LPS、500 ng/m L LPS均能显著提高BEAS-2B细胞中PERK和IRE1α蛋白的表达(P<0.01),而不影响ATF6蛋白的表达(P>0.05)。时效关系结果显示,200ng/m L LPS诱导BEAS-2B细胞0 h、0.5 h、1 h、6 h、12 h和24 h。与空白对照组比较,200 ng/m L LPS诱导6 h,PERK蛋白表达上调(P<0.05),BIP和真核翻译起始因子2α(translational initiation factor 2αin eukaryotes,e IF2α)蛋白在LPS诱导12 h后表达明显上调(P<0.05),并具有时间依赖性,而磷酸化PERK只有在LPS诱导6 h时表达升高(P<0.05)。结果提示,适当LPS刺激BEAS-2B可成功构建体外ERS模型,UPR的两条信号通路PERK、IRE1α选择性活化。试验二PRMT1在PERK-e IF2α信号通路中的作用及机制研究本试验通过RT-q PCR、Western Blot方法研究LPS诱导BEAS-2B细胞蛋白质精氨酸N-甲基转移酶1(protein arginine N-methyltransferase 1,PRMT1)表达的量效关系和时效关系、细胞免疫荧光法检测PRMT1亚细胞定位情况,初步探讨PRMT1对BEAS-2B细胞ERS的影响;利用敲低和过表达PRMT1方法,研究PRMT1在PERK-e IF2α信号通路中的作用机制。剂量-反应关系结果显示,添加50 ng/m L LPS、100 ng/m L LPS、200ng/m L LPS和500 ng/m L LPS均降低BEAS-2B细胞PRMT1转录水平的表达(P<0.001);200 ng/m L LPS和500 ng/m L LPS降低PRMT1蛋白水平的表达(P<0.05)。时间-效应关系结果显示,在200 ng/m L LPS刺激BEAS-2B细胞1 h后,PRMT1蛋白的表达低于对照组(P<0.05),并且具有时间效应。Western Blot结果显示:10μM精氨酸N-甲基转移酶抑制剂1(arginine N-methyltransferase inhibitor-1,AMI-1)处理细胞12 h可抑制PRMT1蛋白水平的表达(P<0.05),但是对磷酸化的PERK无影响(P>0.05);过表达PRMT1质粒转染BEAS-2B细胞72 h,PRMT1转录水平和蛋白水平的表达均显著高于空载质粒组(P<0.01),PERK-e IF2α信号通路蛋白磷酸化PERK(P<0.01)及其下游e IF2α蛋白表达降低(P<0.05)。结果提示,PRMT1在PERK-e IF2α信号通路上游发挥作用。试验三APS对LPS诱导的BEAS-2B细胞体外抗炎作用的研究为了阐明LPS诱导的ERS在BEAS-2B细胞中的功能意义,APS对LPS诱导的体外BEAS-2B细胞的抗炎活性。该研究首先检测LPS浓度对BEAS-2B细胞数量和增殖能力的影响。因此,确定LPS添加剂量用于随后的抗炎试验研究。其次,RT-q PCR检测LPS、APS、LPS+APS组炎性细胞因子的表达情况。MTT结果显示,添加1μg/m L LPS降低BEAS-2B细胞的OD570值(P<0.001),而添加50 ng/m L LPS、100ng/m L LPS、200 ng/m L LPS和500 ng/m L LPS刺激BEAS-2B细胞24 h均无显著影响(P>0.05),因此选择200 ng/m L和500 ng/m L的LPS用于后续抗炎研究。转录水平结果表明,添加200 ng/m L LPS和500 ng/m L LPS可提高BEAS-2B细胞中TNF-αm RNA表达(P<0.05),200 ng/m L LPS+200μg/m L APS和500 ng/m L LPS+200μg/m L APS则无显著影响(P>0.05);添加200 ng/m L LPS和500 ng/m L LPS可使IL-1βm RNA表达显著提高(P<0.01),200 ng/m L LPS+200μg/m L APS添加无显著变化(P>0.05),而与500 ng/m L LPS处理组相比,500 ng/m L LPS+200μg/m L APS显著降低炎症因子IL-1β的表达(P<0.05)。结果表明:APS对LPS刺激BEAS-2B细胞具有抗炎保护作用,此作用可通过降低细胞炎症因子表达实现。综上,表观遗传修饰酶PRMT1参与LPS诱导的BEAS-2B细胞内质网炎症反应,且体外添加APS能够缓解此反应。
Abstract
ji yao hua yang shi mi du de zeng jia jia chong le jia qin hu xi ji tong ji bing ya li 。jia qin fei sun shang shi yan chong de hu xi ji tong bing zheng ,tong chang you huan jing bian hua 、ying ji 、bing yuan gan ran deng duo chong yin su yin qi 。ji fa bing ji zhi shou duo xi bao qi 、xi bao yin zi he xin hao tong lu diao kong 。nei zhi wang ying ji xin hao tong lu shi jie dao qi dao jie gou xi bao ming yun de chong yao tu jing ,ou bie yu ji ta liang tiao diao kong xi bao ming yun de xian li ti tu jing he si wang shou ti tu jing 。biao guan wei chuan xue can yu duo chong sheng ming huo dong (xi bao sheng chang 、fen hua 、diao wang ),zhi zai cong fen zi shui ping jie shi lin chuang zheng zhuang 。ben yan jiu zhong ,wo men li yong zhi duo tang (lipopolysaccharide,LPS)you dao ren zheng chang fei shang pi xi bao (BEAS-2B)gou jian ti wai nei zhi wang ying ji (endoplasmic retieulumstress,ERS)mo xing ,fen xi biao guan wei chuan xiu shi mei de biao da bian hua ji ji sheng wu xue gong neng ,bing tan tao huang qi duo tang (astragalus polysaccharides,APS)dui LPSci ji de cu yan xing xi bao yin zi de diao kong zuo yong ,yi ji wei ying yang biao guan wei chuan diao kong fei bu yan zheng fan ying dian ding li lun ji chu 。shi yan yi LPSyou dao BEAS-2Bxi bao nei zhi wang ying ji mo xing de gou jian ben shi yan shou xian jian ce LPSci ji BEAS-2Bxi bao ERSfa sheng biao zhi xing dan bai mian yi qiu dan bai chong lian jie ge dan bai (immunoglobulin heavy chain binding protein,BIP)de biao da qing kuang ,gou jian ERSmo xing ;ji ci ,fen xi wei she die dan bai fan ying (unfolded protein response,UPR)xin hao tong lu dan bai ji chun xu yao mei 1α(inositol-requiring enzyme 1α,IRE1α)、dan bai ji mei RNAyang nei zhi wang ji mei (the kinase PKR-like ER-resident kinase,PERK)he ji huo zhuai lu yin zi 6(activating transcription factor 6,ATF6)de biao da qing kuang ,tan tao LPSdui BEAS-2Bxi bao ERSde ying xiang ji zuo yong ji zhi 。dan bai shui ping yan jiu LPStian jia dui BEAS-2Bxi bao ERSxiang guan dan bai biao da de ji liang -xiao ying guan ji he shi jian -xiao ying guan ji 。ji liang -xiao ying guan ji de jie guo xian shi ,100 ng/m L LPS、200 ng/m L LPShe 500 ng/m L LPSyi ji liang yi lai xing fang shi zeng jia BEAS-2Bxi bao zhong BIPdan bai de biao da (P<0.01)。tian jia 200ng/m L LPS、500 ng/m L LPSjun neng xian zhe di gao BEAS-2Bxi bao zhong PERKhe IRE1αdan bai de biao da (P<0.01),er bu ying xiang ATF6dan bai de biao da (P>0.05)。shi xiao guan ji jie guo xian shi ,200ng/m L LPSyou dao BEAS-2Bxi bao 0 h、0.5 h、1 h、6 h、12 hhe 24 h。yu kong bai dui zhao zu bi jiao ,200 ng/m L LPSyou dao 6 h,PERKdan bai biao da shang diao (P<0.05),BIPhe zhen he fan yi qi shi yin zi 2α(translational initiation factor 2αin eukaryotes,e IF2α)dan bai zai LPSyou dao 12 hhou biao da ming xian shang diao (P<0.05),bing ju you shi jian yi lai xing ,er lin suan hua PERKzhi you zai LPSyou dao 6 hshi biao da sheng gao (P<0.05)。jie guo di shi ,kuo dang LPSci ji BEAS-2Bke cheng gong gou jian ti wai ERSmo xing ,UPRde liang tiao xin hao tong lu PERK、IRE1αshua ze xing huo hua 。shi yan er PRMT1zai PERK-e IF2αxin hao tong lu zhong de zuo yong ji ji zhi yan jiu ben shi yan tong guo RT-q PCR、Western Blotfang fa yan jiu LPSyou dao BEAS-2Bxi bao dan bai zhi jing an suan N-jia ji zhuai yi mei 1(protein arginine N-methyltransferase 1,PRMT1)biao da de liang xiao guan ji he shi xiao guan ji 、xi bao mian yi ying guang fa jian ce PRMT1ya xi bao ding wei qing kuang ,chu bu tan tao PRMT1dui BEAS-2Bxi bao ERSde ying xiang ;li yong qiao di he guo biao da PRMT1fang fa ,yan jiu PRMT1zai PERK-e IF2αxin hao tong lu zhong de zuo yong ji zhi 。ji liang -fan ying guan ji jie guo xian shi ,tian jia 50 ng/m L LPS、100 ng/m L LPS、200ng/m L LPShe 500 ng/m L LPSjun jiang di BEAS-2Bxi bao PRMT1zhuai lu shui ping de biao da (P<0.001);200 ng/m L LPShe 500 ng/m L LPSjiang di PRMT1dan bai shui ping de biao da (P<0.05)。shi jian -xiao ying guan ji jie guo xian shi ,zai 200 ng/m L LPSci ji BEAS-2Bxi bao 1 hhou ,PRMT1dan bai de biao da di yu dui zhao zu (P<0.05),bing ju ju you shi jian xiao ying 。Western Blotjie guo xian shi :10μMjing an suan N-jia ji zhuai yi mei yi zhi ji 1(arginine N-methyltransferase inhibitor-1,AMI-1)chu li xi bao 12 hke yi zhi PRMT1dan bai shui ping de biao da (P<0.05),dan shi dui lin suan hua de PERKmo ying xiang (P>0.05);guo biao da PRMT1zhi li zhuai ran BEAS-2Bxi bao 72 h,PRMT1zhuai lu shui ping he dan bai shui ping de biao da jun xian zhe gao yu kong zai zhi li zu (P<0.01),PERK-e IF2αxin hao tong lu dan bai lin suan hua PERK(P<0.01)ji ji xia you e IF2αdan bai biao da jiang di (P<0.05)。jie guo di shi ,PRMT1zai PERK-e IF2αxin hao tong lu shang you fa hui zuo yong 。shi yan san APSdui LPSyou dao de BEAS-2Bxi bao ti wai kang yan zuo yong de yan jiu wei le chan ming LPSyou dao de ERSzai BEAS-2Bxi bao zhong de gong neng yi yi ,APSdui LPSyou dao de ti wai BEAS-2Bxi bao de kang yan huo xing 。gai yan jiu shou xian jian ce LPSnong du dui BEAS-2Bxi bao shu liang he zeng shi neng li de ying xiang 。yin ci ,que ding LPStian jia ji liang yong yu sui hou de kang yan shi yan yan jiu 。ji ci ,RT-q PCRjian ce LPS、APS、LPS+APSzu yan xing xi bao yin zi de biao da qing kuang 。MTTjie guo xian shi ,tian jia 1μg/m L LPSjiang di BEAS-2Bxi bao de OD570zhi (P<0.001),er tian jia 50 ng/m L LPS、100ng/m L LPS、200 ng/m L LPShe 500 ng/m L LPSci ji BEAS-2Bxi bao 24 hjun mo xian zhe ying xiang (P>0.05),yin ci shua ze 200 ng/m Lhe 500 ng/m Lde LPSyong yu hou xu kang yan yan jiu 。zhuai lu shui ping jie guo biao ming ,tian jia 200 ng/m L LPShe 500 ng/m L LPSke di gao BEAS-2Bxi bao zhong TNF-αm RNAbiao da (P<0.05),200 ng/m L LPS+200μg/m L APShe 500 ng/m L LPS+200μg/m L APSze mo xian zhe ying xiang (P>0.05);tian jia 200 ng/m L LPShe 500 ng/m L LPSke shi IL-1βm RNAbiao da xian zhe di gao (P<0.01),200 ng/m L LPS+200μg/m L APStian jia mo xian zhe bian hua (P>0.05),er yu 500 ng/m L LPSchu li zu xiang bi ,500 ng/m L LPS+200μg/m L APSxian zhe jiang di yan zheng yin zi IL-1βde biao da (P<0.05)。jie guo biao ming :APSdui LPSci ji BEAS-2Bxi bao ju you kang yan bao hu zuo yong ,ci zuo yong ke tong guo jiang di xi bao yan zheng yin zi biao da shi xian 。zeng shang ,biao guan wei chuan xiu shi mei PRMT1can yu LPSyou dao de BEAS-2Bxi bao nei zhi wang yan zheng fan ying ,ju ti wai tian jia APSneng gou huan jie ci fan ying 。
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论文作者分别是来自西北农林科技大学的卢红，发表于刊物西北农林科技大学2019-07-11论文，是一篇关于脂多糖论文,内质网应激论文,蛋白质精氨酸甲基转移酶论文,肺部炎症论文,黄芪多糖论文,西北农林科技大学2019-07-11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，资料来自西北农林科技大学2019-07-11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：脂多糖论文; 内质网应激论文; 蛋白质精氨酸甲基转移酶论文; 肺部炎症论文; 黄芪多糖论文; 西北农林科技大学2019-07-11论文;