本文主要研究内容
作者莫艳秀（2019）在《SP600125诱导鱼类细胞多倍化的分子机制研究》一文中研究指出：前期研究发现化学小分子SP600125能够诱导鱼类细胞发生多倍化并成功获得稳定的同源四倍体鲫鱼细胞系。为进一步了解SP600125诱导鱼类细胞多倍化的分子机制,本研究通过RNA-seq方法获取SP600125处理组和对照组细胞的差异基因表达谱,利用GO和KEGG通路富集分析全面挖掘SP600125诱导细胞多倍化和细胞稳态维持相关的信号调控网络和关键功能基因,旨在揭示SP600125诱导鱼类细胞多倍化的作用机制,以期为SP600125在鱼类发育生物学的研究以及多倍体培育中的应用提供理论依据和技术参考。具体结果如下:1.高通量测序分析SP600125处理细胞全转录组水平的变化采用高通量测序技术,对体外培养的二倍体鲫尾鳍细胞和SP600125处理组细胞进行了转录组测序和比较分析。获得96,997个unigenes并对unigenes进行了GO功能注释和KEGG通路富集分析。比较SP600125处理组与对照组共获得差异表达基因4516个,其中显著上调有2670个,显著下调有1846个。差异基因主要与细胞代谢、应激反应、发育过程以及细胞周期调控相关。结果显示SP600125诱导引起一批细胞增殖调控相关功能基因尤其以细胞周期检测点相关基因表达水平的改变最为突出,表明SP600125对细胞周期检验点的多重调控与其实现鱼类细胞多倍化的诱导密切相关。2.p53信号通路对SP600125诱导细胞多倍化的调控作用基于转录组学分析的结果,进一步鉴定了与细胞周期检测点密切相关的p53信号通路在SP600125诱导细胞多倍化中的作用。根据已建立的SP600125诱导多倍化的间隔循环策略,利用qRT-PCR和Western blot技术检测SP600125处理对p53信号通路相关基因的mRNA和蛋白水平的影响,结果表明SP600125诱导引起p53、Mdm2、p21和Gadd45ɑ等细胞周期调控相关功能基因的mRNA表达水平产生显著上调,这与转录组分析的结果具有一致性;Western blot结果亦表明SP600125处理48h后细胞中的p53、MDM2和p21的蛋白表达明显上调。为进一步验证p53通路在SP600125诱导细胞多倍体中的作用,以p53抑制剂PFT-ɑ孵育细胞,结果显示PFT-ɑ处理可以在一定程度上降低SP600125对细胞周期G2/M期的阻滞和减少多倍体细胞的产生。以上结果表明p53通路通过调控细胞周期发生G2/M期阻滞参与SP600125诱导鱼类细胞多倍化的产生。3.纺锤体检测点（SAC）基因对SP600125诱导细胞多倍化的影响细胞学观察显示经SP600125处理导致细胞停滞于有丝分裂期。转录组学分析和qRT-PCR验证结果均表明SP600125导致细胞CyclinBl/3、Cdc2、Securin以及负责与动点粘附和对染色体分离监控的纺锤体检验点（SAC）相关基因Bub1、Mad2和Cdc20的mRNA水平呈下调性变化。Western blot结果进一步证实SP600125诱导引起细胞中的BUB1、MAD2和CDC20蛋白的表达水平下降。利用腺病毒成功构建了纺锤体检验点（SAC）核心蛋白MAD2的过表达重组载体（pAd-mCMV-MAD2-3-Flag-GFP-pA）。结合qRT-PCR、Western blot技术以及流式细胞仪分析,进一步鉴定了过表达MAD2对SP600125诱导细胞多倍化的影响。结果证实过表达MAD2能明显减弱SP600125间隔循环处理所引起细胞多倍体的发生。以上结果表明经SP600125诱导引起CyclinBl-Cdc2复合物和纺锤体检验点（SAC）活力下降导致有丝分裂滑移和核内有丝分裂共同产生细胞多倍化。4.急性免疫应激参与SP600125胁迫下的细胞内稳态维持调节基于转录组测序分析,二倍体鲫尾鳍细胞经SP600125处理后能够引起大量免疫炎症相关基因表达水平发生显著的改变,其中涉及到参与先天防御、炎症途径和细胞粘附分子相关途径包括促炎细胞因子（如IL-1β、TNF-ɑ和IL-6R）、转录因子信号转导分子（如Stat-1/3和IκB）、细胞粘附分子（如Vcam-1）和整合素（ɑ2β1和ɑMβ2）等。同时SP600125处理导致线粒体不仅在形态和数量发生明显改变而且在Glut1和主要糖酵解酶基因（如Hk1、Pfbfk、Pgam、Eno和Ldh）的mRNA水平显著降低以及线粒体电子传递链基因（如NADH脱氢酶的Nd1、Nd2和Nd5复合体I亚基基因、Adh、Ndufs8、Ndufa4、Uqcr和Sdhd等）的mRNA水平也显著下调。相关研究结果揭示了在SP600125的胁迫下细胞通过应激免疫响应和降低氧化磷酸化水平等措施参与细胞内稳态的维持。结论:（1）转录组学分析表明SP600125对细胞周期检验点的多重调控与其实现鱼类细胞多倍化的诱导密切相关。（2）SP600125诱导增强了p53信号通路的G2/M期阻滞并导致纺锤体检验点（SAC）信号削弱引发的有丝分裂滑移和核内有丝分裂从而实现鱼类细胞多倍化。（3）在SP600125胁迫下细胞通过应激免疫响应和降低氧化磷酸化水平等措施参与细胞内稳态的维持。研究结果为SP600125在鱼类发育生物学研究和生产实践中的应用奠定了理论基础。
Abstract
qian ji yan jiu fa xian hua xue xiao fen zi SP600125neng gou you dao yu lei xi bao fa sheng duo bei hua bing cheng gong huo de wen ding de tong yuan si bei ti ji yu xi bao ji 。wei jin yi bu le jie SP600125you dao yu lei xi bao duo bei hua de fen zi ji zhi ,ben yan jiu tong guo RNA-seqfang fa huo qu SP600125chu li zu he dui zhao zu xi bao de cha yi ji yin biao da pu ,li yong GOhe KEGGtong lu fu ji fen xi quan mian wa jue SP600125you dao xi bao duo bei hua he xi bao wen tai wei chi xiang guan de xin hao diao kong wang lao he guan jian gong neng ji yin ,zhi zai jie shi SP600125you dao yu lei xi bao duo bei hua de zuo yong ji zhi ,yi ji wei SP600125zai yu lei fa yo sheng wu xue de yan jiu yi ji duo bei ti pei yo zhong de ying yong di gong li lun yi ju he ji shu can kao 。ju ti jie guo ru xia :1.gao tong liang ce xu fen xi SP600125chu li xi bao quan zhuai lu zu shui ping de bian hua cai yong gao tong liang ce xu ji shu ,dui ti wai pei yang de er bei ti ji wei qi xi bao he SP600125chu li zu xi bao jin hang le zhuai lu zu ce xu he bi jiao fen xi 。huo de 96,997ge unigenesbing dui unigenesjin hang le GOgong neng zhu shi he KEGGtong lu fu ji fen xi 。bi jiao SP600125chu li zu yu dui zhao zu gong huo de cha yi biao da ji yin 4516ge ,ji zhong xian zhe shang diao you 2670ge ,xian zhe xia diao you 1846ge 。cha yi ji yin zhu yao yu xi bao dai xie 、ying ji fan ying 、fa yo guo cheng yi ji xi bao zhou ji diao kong xiang guan 。jie guo xian shi SP600125you dao yin qi yi pi xi bao zeng shi diao kong xiang guan gong neng ji yin you ji yi xi bao zhou ji jian ce dian xiang guan ji yin biao da shui ping de gai bian zui wei tu chu ,biao ming SP600125dui xi bao zhou ji jian yan dian de duo chong diao kong yu ji shi xian yu lei xi bao duo bei hua de you dao mi qie xiang guan 。2.p53xin hao tong lu dui SP600125you dao xi bao duo bei hua de diao kong zuo yong ji yu zhuai lu zu xue fen xi de jie guo ,jin yi bu jian ding le yu xi bao zhou ji jian ce dian mi qie xiang guan de p53xin hao tong lu zai SP600125you dao xi bao duo bei hua zhong de zuo yong 。gen ju yi jian li de SP600125you dao duo bei hua de jian ge xun huan ce lve ,li yong qRT-PCRhe Western blotji shu jian ce SP600125chu li dui p53xin hao tong lu xiang guan ji yin de mRNAhe dan bai shui ping de ying xiang ,jie guo biao ming SP600125you dao yin qi p53、Mdm2、p21he Gadd45兺 deng xi bao zhou ji diao kong xiang guan gong neng ji yin de mRNAbiao da shui ping chan sheng xian zhe shang diao ,zhe yu zhuai lu zu fen xi de jie guo ju you yi zhi xing ;Western blotjie guo yi biao ming SP600125chu li 48hhou xi bao zhong de p53、MDM2he p21de dan bai biao da ming xian shang diao 。wei jin yi bu yan zheng p53tong lu zai SP600125you dao xi bao duo bei ti zhong de zuo yong ,yi p53yi zhi ji PFT-兺 fu yo xi bao ,jie guo xian shi PFT-兺 chu li ke yi zai yi ding cheng du shang jiang di SP600125dui xi bao zhou ji G2/Mji de zu zhi he jian shao duo bei ti xi bao de chan sheng 。yi shang jie guo biao ming p53tong lu tong guo diao kong xi bao zhou ji fa sheng G2/Mji zu zhi can yu SP600125you dao yu lei xi bao duo bei hua de chan sheng 。3.fang chui ti jian ce dian （SAC）ji yin dui SP600125you dao xi bao duo bei hua de ying xiang xi bao xue guan cha xian shi jing SP600125chu li dao zhi xi bao ting zhi yu you si fen lie ji 。zhuai lu zu xue fen xi he qRT-PCRyan zheng jie guo jun biao ming SP600125dao zhi xi bao CyclinBl/3、Cdc2、Securinyi ji fu ze yu dong dian nian fu he dui ran se ti fen li jian kong de fang chui ti jian yan dian （SAC）xiang guan ji yin Bub1、Mad2he Cdc20de mRNAshui ping cheng xia diao xing bian hua 。Western blotjie guo jin yi bu zheng shi SP600125you dao yin qi xi bao zhong de BUB1、MAD2he CDC20dan bai de biao da shui ping xia jiang 。li yong xian bing du cheng gong gou jian le fang chui ti jian yan dian （SAC）he xin dan bai MAD2de guo biao da chong zu zai ti （pAd-mCMV-MAD2-3-Flag-GFP-pA）。jie ge qRT-PCR、Western blotji shu yi ji liu shi xi bao yi fen xi ,jin yi bu jian ding le guo biao da MAD2dui SP600125you dao xi bao duo bei hua de ying xiang 。jie guo zheng shi guo biao da MAD2neng ming xian jian ruo SP600125jian ge xun huan chu li suo yin qi xi bao duo bei ti de fa sheng 。yi shang jie guo biao ming jing SP600125you dao yin qi CyclinBl-Cdc2fu ge wu he fang chui ti jian yan dian （SAC）huo li xia jiang dao zhi you si fen lie hua yi he he nei you si fen lie gong tong chan sheng xi bao duo bei hua 。4.ji xing mian yi ying ji can yu SP600125xie pai xia de xi bao nei wen tai wei chi diao jie ji yu zhuai lu zu ce xu fen xi ,er bei ti ji wei qi xi bao jing SP600125chu li hou neng gou yin qi da liang mian yi yan zheng xiang guan ji yin biao da shui ping fa sheng xian zhe de gai bian ,ji zhong she ji dao can yu xian tian fang yu 、yan zheng tu jing he xi bao nian fu fen zi xiang guan tu jing bao gua cu yan xi bao yin zi （ru IL-1β、TNF-兺 he IL-6R）、zhuai lu yin zi xin hao zhuai dao fen zi （ru Stat-1/3he IκB）、xi bao nian fu fen zi （ru Vcam-1）he zheng ge su （兺 2β1he 兺 Mβ2）deng 。tong shi SP600125chu li dao zhi xian li ti bu jin zai xing tai he shu liang fa sheng ming xian gai bian er ju zai Glut1he zhu yao tang jiao jie mei ji yin （ru Hk1、Pfbfk、Pgam、Enohe Ldh）de mRNAshui ping xian zhe jiang di yi ji xian li ti dian zi chuan di lian ji yin （ru NADHtuo qing mei de Nd1、Nd2he Nd5fu ge ti Iya ji ji yin 、Adh、Ndufs8、Ndufa4、Uqcrhe Sdhddeng ）de mRNAshui ping ye xian zhe xia diao 。xiang guan yan jiu jie guo jie shi le zai SP600125de xie pai xia xi bao tong guo ying ji mian yi xiang ying he jiang di yang hua lin suan hua shui ping deng cuo shi can yu xi bao nei wen tai de wei chi 。jie lun :（1）zhuai lu zu xue fen xi biao ming SP600125dui xi bao zhou ji jian yan dian de duo chong diao kong yu ji shi xian yu lei xi bao duo bei hua de you dao mi qie xiang guan 。（2）SP600125you dao zeng jiang le p53xin hao tong lu de G2/Mji zu zhi bing dao zhi fang chui ti jian yan dian （SAC）xin hao xiao ruo yin fa de you si fen lie hua yi he he nei you si fen lie cong er shi xian yu lei xi bao duo bei hua 。（3）zai SP600125xie pai xia xi bao tong guo ying ji mian yi xiang ying he jiang di yang hua lin suan hua shui ping deng cuo shi can yu xi bao nei wen tai de wei chi 。yan jiu jie guo wei SP600125zai yu lei fa yo sheng wu xue yan jiu he sheng chan shi jian zhong de ying yong dian ding le li lun ji chu 。
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论文作者分别是来自湖南师范大学的莫艳秀，发表于刊物湖南师范大学2019-10-31论文，是一篇关于高通量测序论文,细胞多倍化论文,细胞周期调节论文,湖南师范大学2019-10-31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，资料来自湖南师范大学2019-10-31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：高通量测序论文; 细胞多倍化论文; 细胞周期调节论文; 湖南师范大学2019-10-31论文;