本文主要研究内容
作者陈梦豪（2019）在《H2S暴露通过lncRNA3037/miR-15a调控BCL2与A20诱导鸡气管细胞凋亡与程序性坏死的研究》一文中研究指出：硫化氢（H2S）是一种危险的空气污染物,随着工业化的不断发展,空气中H2S污染引起的危害逐渐引起人们的重视。H2S可对机体的呼吸系统、心血管系统及神经系统等多个系统的组织器官产生毒性效应。细胞凋亡与程序性坏死是外源性毒物诱导细胞死亡的主要形式,这两种细胞死亡模式在呼吸道疾病中起重要作用。MicroRNA（miRNA）作为一种非编码RNA,可以通过靶向调控其目的基因转录后表达介导体内多种生物反应,对于调控细胞执行程序性死亡发挥重要作用。近年来研究发现miRNA在一些环境污染物中毒和疾病中发挥重要调控作用,miRNA逐渐被认可作为一种环境污染物与某些疾病的联系的潜在生物标志物。长链非编码RNA（lncRNA）可在细胞质中与miRNA结合,作为分子海绵调控miRNA发挥功能。目前,lncRNA是否参与H2S诱导的呼吸道损伤,且功能如何还未见报道。因此,本试验以肉鸡为研究对象,通过在环境控制仓中持续通入H2S（0-3周龄4 mg/m3,4-6周龄20 mg/m3）气体来建立体内H2S暴露模型,添加H2S外源性供体NaHS建立体外鸡肝癌细胞（LMH）H2S暴露模型,应用透射电镜、扫描电镜、转录组学检测技术、免疫印迹、实时荧光定量PCR技术、流式细胞术、生物信息学方法等,检测气管组织形态学变化、lncRNA表达谱、细胞凋亡与程序性坏死,以及分析筛选与细胞凋亡和程序性坏死相关信号转导通路、构建lncRNA-miRNA-mRNA调控网络并进行验证,旨在证实H2S暴露引起的气管组织细胞死亡的类型,阐明lncRNA在其中的作用机制。主要研究结果如下:（1）病理组织学观察结果显示,H2S暴露后引起了鸡气管上皮细胞脱落,其中纤毛细胞缺失严重,在病灶处有大量炎性细胞浸润。扫描电镜结果显示气管上皮纤毛缺失、凌乱,大量粘液附着在纤毛表面。透射电镜观察结果显示,H2S暴露组中,细胞膜、细胞核膜连续性破坏;染色质凝聚、边聚;线粒体嵴肿胀、破裂、空泡化;粗面内质网脱颗粒、断裂。表现为典型的细胞坏死和细胞凋亡特征,说明H2S暴露能够引起气管组织炎性损伤、细胞凋亡及坏死的发生。（2）H2S暴露引起鸡气管组织中6个lncRNAs表达上调,10个lncRNAs表达下调（log2fold change>0.585或<-0.585,FDR<0.05）。通过qRT-PCR技术对部分转录本进行验证,证实了转录测序技术的可靠性,表明H2S暴露能够影响鸡气管组织lncRNA的表达。对下调的前五个lncRNAs进行细胞内定位,发现lncRNA3037是定位于细胞质中表达下调最大的lncRNA。（3）通过生物信息学技术,预测lncRNA3037能够与miR-15a靶向结合,并且miR-15a在H2S暴露组鸡气管组织中的表达量显著高于对照组,暗示lncRNA3037可能作为miR-15a的分子海绵。双荧光素酶报告系统检测证实了lncRNA3037能够靶向结合miR-15a。通过lncRNA3037和miR-15a过表达与敲低,证明lncRNA3037与miR-15a表达均可负向调控。另外,敲低lncRNA3037或过表达miR-15a可抑制miR-15a靶基因BCL2与A20的表达,敲低miR-15a能够逆转敲低lncRNA3037对与BCL2与A20的抑制作用。上述结果表明lncRNA3037能够作为miR-15a的分子海绵调控miR-15a靶基因A20与BCL2的表达。（4）H2S暴露诱导气管组织中程序性坏死相关蛋白RIPK1、RIPK3和p-MLKL表达升高,抗程序性坏死因子A20表达降低,凋亡诱导因子BAX与caspase 3升高,抗凋亡因子BCL2表达下调。体外试验结果显示,H2S暴露后LMH细胞活性以浓度依赖性方式降低,细胞凋亡率与坏死率均增高,程序性坏死和凋亡相关蛋白也呈相应的表达模式。证实了H2S暴露引起鸡气管组织和LMH细胞程序性坏死和凋亡的发生。在建立鸡LMH细胞miR-15a过表达/敲低模型的基础上,用程序性坏死抑制剂（Nec-1）、凋亡抑制剂（z-VAD-fmk）、H2S供体NaHS与lncRNA3037的siRNA处理细胞,结果显示miR-15a过表达细胞的程序性坏死率与凋亡率明显升高,并可分别被Nec-1或z-VAD-fmk抑制。另外,敲低miR-15a能够减少NaHS处理或lncRNA3037敲低诱导的细胞凋亡与程序性坏死率。上述结果表明,lncRNA3037的表达降低,导致其不能竞争性吸附miR-15a,增强了miR-15a对靶基因BCL2与A20的抑制作用,进而引起细胞凋亡和程序性坏死发生。综上所述,H2S暴露能够引起鸡气管组织炎性损伤、细胞凋亡与程序性坏死发生,诱导lncRNA表达谱变化,其中定位于细胞质中的lncRNA3037表达降低,miR-15a表达升高,A20与BCL2表达降低。A20与BCL2为miR-15a的靶基因,lncRNA3037能够靶向结合miR-15a而调控BCL2与A20表达。进一步研究证实,H2S暴露抑制lncRNA3037表达,通过miR-15a下调BCL2与A20的表达引起细胞凋亡与程序性坏死的发生。本研究结果不仅为H2S毒理学研究提供科学依据,同时为H2S暴露相关疾病提供可能的预防和治疗依据手段,为畜禽健康养殖提供环境决策依据。
Abstract
liu hua qing （H2S）shi yi chong wei xian de kong qi wu ran wu ,sui zhao gong ye hua de bu duan fa zhan ,kong qi zhong H2Swu ran yin qi de wei hai zhu jian yin qi ren men de chong shi 。H2Ske dui ji ti de hu xi ji tong 、xin xie guan ji tong ji shen jing ji tong deng duo ge ji tong de zu zhi qi guan chan sheng du xing xiao ying 。xi bao diao wang yu cheng xu xing huai si shi wai yuan xing du wu you dao xi bao si wang de zhu yao xing shi ,zhe liang chong xi bao si wang mo shi zai hu xi dao ji bing zhong qi chong yao zuo yong 。MicroRNA（miRNA）zuo wei yi chong fei bian ma RNA,ke yi tong guo ba xiang diao kong ji mu de ji yin zhuai lu hou biao da jie dao ti nei duo chong sheng wu fan ying ,dui yu diao kong xi bao zhi hang cheng xu xing si wang fa hui chong yao zuo yong 。jin nian lai yan jiu fa xian miRNAzai yi xie huan jing wu ran wu zhong du he ji bing zhong fa hui chong yao diao kong zuo yong ,miRNAzhu jian bei ren ke zuo wei yi chong huan jing wu ran wu yu mou xie ji bing de lian ji de qian zai sheng wu biao zhi wu 。chang lian fei bian ma RNA（lncRNA）ke zai xi bao zhi zhong yu miRNAjie ge ,zuo wei fen zi hai mian diao kong miRNAfa hui gong neng 。mu qian ,lncRNAshi fou can yu H2Syou dao de hu xi dao sun shang ,ju gong neng ru he hai wei jian bao dao 。yin ci ,ben shi yan yi rou ji wei yan jiu dui xiang ,tong guo zai huan jing kong zhi cang zhong chi xu tong ru H2S（0-3zhou ling 4 mg/m3,4-6zhou ling 20 mg/m3）qi ti lai jian li ti nei H2Sbao lou mo xing ,tian jia H2Swai yuan xing gong ti NaHSjian li ti wai ji gan ai xi bao （LMH）H2Sbao lou mo xing ,ying yong tou she dian jing 、sao miao dian jing 、zhuai lu zu xue jian ce ji shu 、mian yi yin ji 、shi shi ying guang ding liang PCRji shu 、liu shi xi bao shu 、sheng wu xin xi xue fang fa deng ,jian ce qi guan zu zhi xing tai xue bian hua 、lncRNAbiao da pu 、xi bao diao wang yu cheng xu xing huai si ,yi ji fen xi shai shua yu xi bao diao wang he cheng xu xing huai si xiang guan xin hao zhuai dao tong lu 、gou jian lncRNA-miRNA-mRNAdiao kong wang lao bing jin hang yan zheng ,zhi zai zheng shi H2Sbao lou yin qi de qi guan zu zhi xi bao si wang de lei xing ,chan ming lncRNAzai ji zhong de zuo yong ji zhi 。zhu yao yan jiu jie guo ru xia :（1）bing li zu zhi xue guan cha jie guo xian shi ,H2Sbao lou hou yin qi le ji qi guan shang pi xi bao tuo la ,ji zhong qian mao xi bao que shi yan chong ,zai bing zao chu you da liang yan xing xi bao jin run 。sao miao dian jing jie guo xian shi qi guan shang pi qian mao que shi 、ling luan ,da liang nian ye fu zhao zai qian mao biao mian 。tou she dian jing guan cha jie guo xian shi ,H2Sbao lou zu zhong ,xi bao mo 、xi bao he mo lian xu xing po huai ;ran se zhi ning ju 、bian ju ;xian li ti ji zhong zhang 、po lie 、kong pao hua ;cu mian nei zhi wang tuo ke li 、duan lie 。biao xian wei dian xing de xi bao huai si he xi bao diao wang te zheng ,shui ming H2Sbao lou neng gou yin qi qi guan zu zhi yan xing sun shang 、xi bao diao wang ji huai si de fa sheng 。（2）H2Sbao lou yin qi ji qi guan zu zhi zhong 6ge lncRNAsbiao da shang diao ,10ge lncRNAsbiao da xia diao （log2fold change>0.585huo <-0.585,FDR<0.05）。tong guo qRT-PCRji shu dui bu fen zhuai lu ben jin hang yan zheng ,zheng shi le zhuai lu ce xu ji shu de ke kao xing ,biao ming H2Sbao lou neng gou ying xiang ji qi guan zu zhi lncRNAde biao da 。dui xia diao de qian wu ge lncRNAsjin hang xi bao nei ding wei ,fa xian lncRNA3037shi ding wei yu xi bao zhi zhong biao da xia diao zui da de lncRNA。（3）tong guo sheng wu xin xi xue ji shu ,yu ce lncRNA3037neng gou yu miR-15aba xiang jie ge ,bing ju miR-15azai H2Sbao lou zu ji qi guan zu zhi zhong de biao da liang xian zhe gao yu dui zhao zu ,an shi lncRNA3037ke neng zuo wei miR-15ade fen zi hai mian 。shuang ying guang su mei bao gao ji tong jian ce zheng shi le lncRNA3037neng gou ba xiang jie ge miR-15a。tong guo lncRNA3037he miR-15aguo biao da yu qiao di ,zheng ming lncRNA3037yu miR-15abiao da jun ke fu xiang diao kong 。ling wai ,qiao di lncRNA3037huo guo biao da miR-15ake yi zhi miR-15aba ji yin BCL2yu A20de biao da ,qiao di miR-15aneng gou ni zhuai qiao di lncRNA3037dui yu BCL2yu A20de yi zhi zuo yong 。shang shu jie guo biao ming lncRNA3037neng gou zuo wei miR-15ade fen zi hai mian diao kong miR-15aba ji yin A20yu BCL2de biao da 。（4）H2Sbao lou you dao qi guan zu zhi zhong cheng xu xing huai si xiang guan dan bai RIPK1、RIPK3he p-MLKLbiao da sheng gao ,kang cheng xu xing huai si yin zi A20biao da jiang di ,diao wang you dao yin zi BAXyu caspase 3sheng gao ,kang diao wang yin zi BCL2biao da xia diao 。ti wai shi yan jie guo xian shi ,H2Sbao lou hou LMHxi bao huo xing yi nong du yi lai xing fang shi jiang di ,xi bao diao wang lv yu huai si lv jun zeng gao ,cheng xu xing huai si he diao wang xiang guan dan bai ye cheng xiang ying de biao da mo shi 。zheng shi le H2Sbao lou yin qi ji qi guan zu zhi he LMHxi bao cheng xu xing huai si he diao wang de fa sheng 。zai jian li ji LMHxi bao miR-15aguo biao da /qiao di mo xing de ji chu shang ,yong cheng xu xing huai si yi zhi ji （Nec-1）、diao wang yi zhi ji （z-VAD-fmk）、H2Sgong ti NaHSyu lncRNA3037de siRNAchu li xi bao ,jie guo xian shi miR-15aguo biao da xi bao de cheng xu xing huai si lv yu diao wang lv ming xian sheng gao ,bing ke fen bie bei Nec-1huo z-VAD-fmkyi zhi 。ling wai ,qiao di miR-15aneng gou jian shao NaHSchu li huo lncRNA3037qiao di you dao de xi bao diao wang yu cheng xu xing huai si lv 。shang shu jie guo biao ming ,lncRNA3037de biao da jiang di ,dao zhi ji bu neng jing zheng xing xi fu miR-15a,zeng jiang le miR-15adui ba ji yin BCL2yu A20de yi zhi zuo yong ,jin er yin qi xi bao diao wang he cheng xu xing huai si fa sheng 。zeng shang suo shu ,H2Sbao lou neng gou yin qi ji qi guan zu zhi yan xing sun shang 、xi bao diao wang yu cheng xu xing huai si fa sheng ,you dao lncRNAbiao da pu bian hua ,ji zhong ding wei yu xi bao zhi zhong de lncRNA3037biao da jiang di ,miR-15abiao da sheng gao ,A20yu BCL2biao da jiang di 。A20yu BCL2wei miR-15ade ba ji yin ,lncRNA3037neng gou ba xiang jie ge miR-15aer diao kong BCL2yu A20biao da 。jin yi bu yan jiu zheng shi ,H2Sbao lou yi zhi lncRNA3037biao da ,tong guo miR-15axia diao BCL2yu A20de biao da yin qi xi bao diao wang yu cheng xu xing huai si de fa sheng 。ben yan jiu jie guo bu jin wei H2Sdu li xue yan jiu di gong ke xue yi ju ,tong shi wei H2Sbao lou xiang guan ji bing di gong ke neng de yu fang he zhi liao yi ju shou duan ,wei chu qin jian kang yang shi di gong huan jing jue ce yi ju 。
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论文作者分别是来自东北农业大学的陈梦豪，发表于刊物东北农业大学2019-08-27论文，是一篇关于硫化氢论文,细胞凋亡论文,程序性坏死论文,东北农业大学2019-08-27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，资料来自东北农业大学2019-08-27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：硫化氢论文; 细胞凋亡论文; 程序性坏死论文; 东北农业大学2019-08-27论文;