本文主要研究内容
作者李丹阳（2019）在《EZH2的一种新型异构体在生物体内的分布和功能研究》一文中研究指出：可变剪接指同一基因在转录后,不同外显子的剪接形式使得同一基因的表达产物有着不同的剪接异构体,常对应着不同的功能。EZH2作为表观调控元件PRC2复合体的重要组成部分,参与细胞生命活动的调节。EZH2的剪接异构体在细胞内有着多种形式,一种被人们忽视的剪接异构体形式EZH2-X9在SD生物体内有着广泛表达,具体功能尚不明确。目的:1.分离鉴定出EZH2-X9型剪接异构体,对核酸序列、氨基酸序列进行比对分析;2.探究EZH2-X9型剪接异构体在细胞和SD大鼠中的表达分布以及随着个体发育过程中不同组织中表达量的变化;3.初步探究EZH2-X9型剪接异构体在细胞中与普通的全长EZH2相比功能上所具有的异同点。方法:1.在PC12细胞和SD大鼠中,通过PCR技术和基因测序寻找EZH2-X9剪接异构体的核酸表达,结合MUSCLE和BLAST对核酸及氨基酸序列进行分析;2.通过非放射性原位杂交技术(FISH)、免疫细胞化学技术(IF)、实时定量基因扩增荧光检测(q-PCR)以及蛋白质印记(WB)研究EZH2-X9剪接异构体在细胞内的表达以及分布;在SD大鼠中,通过特异性引物的设计和使用,q-PCR对不同组织中的mRNA表达分布进行研究,并利用WB检测蛋白表达的同步性。3.在未分化的PC12细胞中,结合基因工程的手段,通过荧光显微镜观察分析并统计细胞的变化,探究EZH2-X9剪接异构体对NGF诱导的细胞分化过程中所起到的具体功能影响。4.在PC12细胞内,通过降解同源序列靶基因mRNA的基因敲低(KD)方式,分别阻止普通EZH2-WT和EZH2-X9剪接异构体的表达,结合WB和蛋白质免疫共沉淀(IP)探究EZH2-WT和EZH2-X9剪接异构体在细胞内的功能差异性。结果:1.EZH2-X9在SD大鼠体内广泛分布,在细胞水平上EZH2-X9剪接异构体主要分布于细胞核内。2.在SD大鼠体内,EZH2-WT和EZH2-X9在不同组织中的表达量有着一定的差异性,脑中表达量和肺、心、肝相比较高;二者在同一组织中随着个体发育时间的增长而衰减,且蛋白表达量同步。3.在PC12细胞中分别过表达和KD二者后,在mRNA水平上发现二者有着相互影响;EZH2-X9对于组蛋白H3第27位赖氨酸的甲基化作用没有显著的影响,IP实验发现EZH2-X9能和EZH2-WT竞争结合EED,组成PRC2复合体;4.EZH2-WT和EZH2-X9对与细胞分化都有着重要的功能,在NGF诱导下未分化PC12细胞分化过程中,在KD-EZH2-X9时细胞的突起生长总长度、突起生长平均长度、突起生长数目、NOI%相比KD-EZH2-WT都有着更为显著的降低;KD-EZH2-X9会提高UIR1、MapLC3和GSK的表达。结论:1.EZH2-X9主要存在于SD大鼠的脑中,呈发育期差异表达;2.EZH2-X9和和EZH2-WT竞争结合EED,组成PRC2复合体却不能介导H3K27me3;3.EZH2-X9和EZH2-WT通过调控不同的靶基因和分子途径调节细胞生长。
Abstract
ke bian jian jie zhi tong yi ji yin zai zhuai lu hou ,bu tong wai xian zi de jian jie xing shi shi de tong yi ji yin de biao da chan wu you zhao bu tong de jian jie yi gou ti ,chang dui ying zhao bu tong de gong neng 。EZH2zuo wei biao guan diao kong yuan jian PRC2fu ge ti de chong yao zu cheng bu fen ,can yu xi bao sheng ming huo dong de diao jie 。EZH2de jian jie yi gou ti zai xi bao nei you zhao duo chong xing shi ,yi chong bei ren men hu shi de jian jie yi gou ti xing shi EZH2-X9zai SDsheng wu ti nei you zhao an fan biao da ,ju ti gong neng shang bu ming que 。mu de :1.fen li jian ding chu EZH2-X9xing jian jie yi gou ti ,dui he suan xu lie 、an ji suan xu lie jin hang bi dui fen xi ;2.tan jiu EZH2-X9xing jian jie yi gou ti zai xi bao he SDda shu zhong de biao da fen bu yi ji sui zhao ge ti fa yo guo cheng zhong bu tong zu zhi zhong biao da liang de bian hua ;3.chu bu tan jiu EZH2-X9xing jian jie yi gou ti zai xi bao zhong yu pu tong de quan chang EZH2xiang bi gong neng shang suo ju you de yi tong dian 。fang fa :1.zai PC12xi bao he SDda shu zhong ,tong guo PCRji shu he ji yin ce xu xun zhao EZH2-X9jian jie yi gou ti de he suan biao da ,jie ge MUSCLEhe BLASTdui he suan ji an ji suan xu lie jin hang fen xi ;2.tong guo fei fang she xing yuan wei za jiao ji shu (FISH)、mian yi xi bao hua xue ji shu (IF)、shi shi ding liang ji yin kuo zeng ying guang jian ce (q-PCR)yi ji dan bai zhi yin ji (WB)yan jiu EZH2-X9jian jie yi gou ti zai xi bao nei de biao da yi ji fen bu ;zai SDda shu zhong ,tong guo te yi xing yin wu de she ji he shi yong ,q-PCRdui bu tong zu zhi zhong de mRNAbiao da fen bu jin hang yan jiu ,bing li yong WBjian ce dan bai biao da de tong bu xing 。3.zai wei fen hua de PC12xi bao zhong ,jie ge ji yin gong cheng de shou duan ,tong guo ying guang xian wei jing guan cha fen xi bing tong ji xi bao de bian hua ,tan jiu EZH2-X9jian jie yi gou ti dui NGFyou dao de xi bao fen hua guo cheng zhong suo qi dao de ju ti gong neng ying xiang 。4.zai PC12xi bao nei ,tong guo jiang jie tong yuan xu lie ba ji yin mRNAde ji yin qiao di (KD)fang shi ,fen bie zu zhi pu tong EZH2-WThe EZH2-X9jian jie yi gou ti de biao da ,jie ge WBhe dan bai zhi mian yi gong chen dian (IP)tan jiu EZH2-WThe EZH2-X9jian jie yi gou ti zai xi bao nei de gong neng cha yi xing 。jie guo :1.EZH2-X9zai SDda shu ti nei an fan fen bu ,zai xi bao shui ping shang EZH2-X9jian jie yi gou ti zhu yao fen bu yu xi bao he nei 。2.zai SDda shu ti nei ,EZH2-WThe EZH2-X9zai bu tong zu zhi zhong de biao da liang you zhao yi ding de cha yi xing ,nao zhong biao da liang he fei 、xin 、gan xiang bi jiao gao ;er zhe zai tong yi zu zhi zhong sui zhao ge ti fa yo shi jian de zeng chang er cui jian ,ju dan bai biao da liang tong bu 。3.zai PC12xi bao zhong fen bie guo biao da he KDer zhe hou ,zai mRNAshui ping shang fa xian er zhe you zhao xiang hu ying xiang ;EZH2-X9dui yu zu dan bai H3di 27wei lai an suan de jia ji hua zuo yong mei you xian zhe de ying xiang ,IPshi yan fa xian EZH2-X9neng he EZH2-WTjing zheng jie ge EED,zu cheng PRC2fu ge ti ;4.EZH2-WThe EZH2-X9dui yu xi bao fen hua dou you zhao chong yao de gong neng ,zai NGFyou dao xia wei fen hua PC12xi bao fen hua guo cheng zhong ,zai KD-EZH2-X9shi xi bao de tu qi sheng chang zong chang du 、tu qi sheng chang ping jun chang du 、tu qi sheng chang shu mu 、NOI%xiang bi KD-EZH2-WTdou you zhao geng wei xian zhe de jiang di ;KD-EZH2-X9hui di gao UIR1、MapLC3he GSKde biao da 。jie lun :1.EZH2-X9zhu yao cun zai yu SDda shu de nao zhong ,cheng fa yo ji cha yi biao da ;2.EZH2-X9he he EZH2-WTjing zheng jie ge EED,zu cheng PRC2fu ge ti que bu neng jie dao H3K27me3;3.EZH2-X9he EZH2-WTtong guo diao kong bu tong de ba ji yin he fen zi tu jing diao jie xi bao sheng chang 。
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论文作者分别是来自合肥工业大学的李丹阳，发表于刊物合肥工业大学2019-09-20论文，是一篇关于可变剪接论文,神经分化论文,合肥工业大学2019-09-20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，资料来自合肥工业大学2019-09-20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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