本文主要研究内容
作者孙福玲（2019）在《lamin基因调控果蝇卵巢生殖干细胞命运的研究》一文中研究指出：干细胞是一类自我更新和多向分化潜能两种特性的细胞群,这些特征决定了其在医学治疗和器官再生方面具有非常广阔的应用前景。生殖干细胞（Germline Stem Cells,GSC）分裂与分化命运决定方面的研究,已成为干细胞与发育生物学领域关注的热点。自摩尔根发现基因的连锁互换定律以来,果蝇（Drosophila melanogaster）一直作为遗传学和发育生物学研究领域重要的模式生物。因具有可靠的干细胞标记和清晰的发育谱系等优点,果蝇卵巢GSC常被作为生殖干细胞研究的优良模型。核纤层蛋白（Lamin）作为一类中间纤维（Intermediate filament,IF）蛋白为细胞核膜提供骨架支撑,同时还参与染色质组织、DNA复制和损伤修复等过程。我们前期的工作发现,与野生型果蝇（oregon）相比,随着时间的推移,lamin基因突变体果蝇卵巢GSC的数量有降低的趋势。为了确证上述结果,进一步探究lamin调控果蝇卵巢GSC命运的机制,我们利用免疫荧光染色、有丝分裂重组和果蝇转基因等技术,研究了lamin基因调控果蝇卵巢GSC命运的初步机制,本研究成果将贡献于干细胞分裂分化调控领域。首先,以野生型果蝇为对照,利用免疫荧光染色技术统计分析了lamin突变体果蝇卵巢GSC在羽化后不同天数的数量变化情况。果蝇卵巢顶端被称为原卵区,正常原卵区一般含有2-3个GSC。结果显示:野生型果蝇羽化后第1天（1d）和第14天（14d）正常原卵区的比例分别为97.7%和96.4%,两周内卵巢GSC数量基本无变化。lamin四组突变体(lam A25/lamA25、lamA25/lamK2、lamA25/lam04643和lamK2/lam04643)果蝇在羽化后第1d正常原卵区的比例分别为78.7%、81.8%、84.8%和88.0%;羽化后第14d分别降至27.9%、36.2%、37.6%和48.1%,表明卵巢GSC发生了显著的丢失,且lamA25/lamA25突变体果蝇卵巢GSC丢失最严重。上述结果表明lamin基因是果蝇卵巢GSC维持所必需的。其次,利用UAS/gal4系统对lamin基因分别实施内源性和外源性RNA干扰（RNA interfering,RNAi）,统计羽化后不同天数（1d、7d和14d）果蝇卵巢GSC数量变化。在内源性RNAi试验中有两个敲低组,分别为lamin基因2号和3号染色体敲低组。结果显示:对照组（未敲低亲本组）果蝇正常原卵区比例在两周内都高达92.0%以上,表明卵巢GSC数量基本没有变化。羽化后1d、7d和14d,lamin基因2号敲低组正常原卵区比例分别为84.1%、36.7%和31.2%;3号敲低组正常原卵区的比例分别为72.1%、54.8%和36.6%。两组RNAi试验结果显示:随着时间推移,内源性敲低lamin导致正常原卵区的比例明显降低,暗示其卵巢GSC数量发生了严重的下降。当外源性敲低lamin基因,实验组统计结果与对照组无明显变化（P>0.05）,表明外源性敲低lamin不影响GSC维持。另外,采用有丝分裂重组技术内源性诱导GSC克隆,分别统计1d、7d和14d标记GSC克隆的数量变化。结果显示:野生型对照组标记的GSC（基因型:FRT40A）的百分比在两周内从1d的43.7%下降到14d的42.1%,GSC数量基本无变化。而三个实验组标记的lamin突变(基因型分别为:FRT40A,lamA25、FRT40A,lamK2与FRT40A,lam04643)的GSC百分比从第1d的43.8%、45.0%和44.0%下降到第14d的15.1%、16.7%和17.3%,两周内分别有65.5%、62.9%和60.7%的GSC发生了丢失,支持lamin内源性地发挥调控GSC维持的功能。上述结果均暗示lamin基因在调控果蝇卵巢GSC维持方面发挥内源性作用。为了进一步确证lamin基因的功能,排除遗传背景的干扰,本课题构建了一系列lamin转基因载体,分别从内源和外源去挽救lamin基因缺失导致的表型。结果显示:内源性补充lamin基因能完全挽救lamin缺失的表型;仅外源性补充lamin基因无法挽救GSC的数量下降,表明lamin基因仅内源性调控果蝇卵巢GSC的维持。此外,借助免疫组织化学染色技术检测Lamin蛋白的表达模式,观察发现,Lamin蛋白在卵巢GSCs中表达量高,且定位于细胞核膜,暗示了该基因在GSC中发挥重要功能。总之,上述结果充分表明lamin基因发挥内源性调控果蝇卵巢GSC命运的功能。为了确证lamin基因表达与GSC维持的相关性,在RNA水平上,利用荧光定量PCR（qPCR）检测了lamin突变体与RNA干扰品系果蝇卵巢中lamin基因的表达。结果显示,与野生型相比,突变体和RNA干扰组中lamin表达量都有明显降低。在蛋白质水平上,利用蛋白免疫印迹（Western Blot）技术检测Lamin蛋白的表达。结果显示:lamin突变体中Lamin蛋白的表达量显著降低。此结果充分表明lamin基因表达下调是导致果蝇卵巢GSCs丢失的直接原因。再次,借助bamP-GFP报告系统研究lamin与已知BMP/Dpp-bam信号通路的关系。结果显示,对照组中GFP阴性标记的GSC数占97.7%,表明bam基因沉默是GSC维持所必需的。在lamin突变的背景下,三组实验组果蝇(bamP-GFP;lamA25/lamA25、bamP-GFP;lamA25/lamK2和bamP-GFP;lamK2/lam04643)卵巢中,分别有18.8%、30.3%和12.5%的GSC中检测到GFP标记的bam的表达,明显高于对照组2.3%比例。该结果表明lamin基因小部分地参与了GSC中bam基因的抑制（位于bam的上游）,同时暗示了lamin基因主要位于bam的下游,或通过平行于BMP/Dpp-bam信号通路来抑制GSC的分化以维持干性。既然lamin突变导致GSC数量丢失,那么缺失的GSCs是否走向凋亡?为了回答此问题,利用TUNEL技术对lamin突变果蝇的卵巢GSCs进行凋亡检测。结果显示,野生型果蝇卵巢GSC凋亡发生率为1.8%,两种lamin突变体卵巢GSC凋亡发生率分别为5.1%和5.3%,表明野生型与突变体果蝇的凋亡发生率无明显差异。此外,利用有丝分裂重组技术获得的内源性lamin突变GSC中也没有发现增加的凋亡率。该结果暗示了lamin突变的GSC提前走向分化而导致丢失。我们的工作加深了对GSC命运决定机制的理解。
Abstract
gan xi bao shi yi lei zi wo geng xin he duo xiang fen hua qian neng liang chong te xing de xi bao qun ,zhe xie te zheng jue ding le ji zai yi xue zhi liao he qi guan zai sheng fang mian ju you fei chang an kuo de ying yong qian jing 。sheng shi gan xi bao （Germline Stem Cells,GSC）fen lie yu fen hua ming yun jue ding fang mian de yan jiu ,yi cheng wei gan xi bao yu fa yo sheng wu xue ling yu guan zhu de re dian 。zi ma er gen fa xian ji yin de lian suo hu huan ding lv yi lai ,guo ying （Drosophila melanogaster）yi zhi zuo wei wei chuan xue he fa yo sheng wu xue yan jiu ling yu chong yao de mo shi sheng wu 。yin ju you ke kao de gan xi bao biao ji he qing xi de fa yo pu ji deng you dian ,guo ying luan chao GSCchang bei zuo wei sheng shi gan xi bao yan jiu de you liang mo xing 。he qian ceng dan bai （Lamin）zuo wei yi lei zhong jian qian wei （Intermediate filament,IF）dan bai wei xi bao he mo di gong gu jia zhi cheng ,tong shi hai can yu ran se zhi zu zhi 、DNAfu zhi he sun shang xiu fu deng guo cheng 。wo men qian ji de gong zuo fa xian ,yu ye sheng xing guo ying （oregon）xiang bi ,sui zhao shi jian de tui yi ,laminji yin tu bian ti guo ying luan chao GSCde shu liang you jiang di de qu shi 。wei le que zheng shang shu jie guo ,jin yi bu tan jiu lamindiao kong guo ying luan chao GSCming yun de ji zhi ,wo men li yong mian yi ying guang ran se 、you si fen lie chong zu he guo ying zhuai ji yin deng ji shu ,yan jiu le laminji yin diao kong guo ying luan chao GSCming yun de chu bu ji zhi ,ben yan jiu cheng guo jiang gong suo yu gan xi bao fen lie fen hua diao kong ling yu 。shou xian ,yi ye sheng xing guo ying wei dui zhao ,li yong mian yi ying guang ran se ji shu tong ji fen xi le lamintu bian ti guo ying luan chao GSCzai yu hua hou bu tong tian shu de shu liang bian hua qing kuang 。guo ying luan chao ding duan bei chen wei yuan luan ou ,zheng chang yuan luan ou yi ban han you 2-3ge GSC。jie guo xian shi :ye sheng xing guo ying yu hua hou di 1tian （1d）he di 14tian （14d）zheng chang yuan luan ou de bi li fen bie wei 97.7%he 96.4%,liang zhou nei luan chao GSCshu liang ji ben mo bian hua 。laminsi zu tu bian ti (lam A25/lamA25、lamA25/lamK2、lamA25/lam04643he lamK2/lam04643)guo ying zai yu hua hou di 1dzheng chang yuan luan ou de bi li fen bie wei 78.7%、81.8%、84.8%he 88.0%;yu hua hou di 14dfen bie jiang zhi 27.9%、36.2%、37.6%he 48.1%,biao ming luan chao GSCfa sheng le xian zhe de diu shi ,ju lamA25/lamA25tu bian ti guo ying luan chao GSCdiu shi zui yan chong 。shang shu jie guo biao ming laminji yin shi guo ying luan chao GSCwei chi suo bi xu de 。ji ci ,li yong UAS/gal4ji tong dui laminji yin fen bie shi shi nei yuan xing he wai yuan xing RNAgan rao （RNA interfering,RNAi）,tong ji yu hua hou bu tong tian shu （1d、7dhe 14d）guo ying luan chao GSCshu liang bian hua 。zai nei yuan xing RNAishi yan zhong you liang ge qiao di zu ,fen bie wei laminji yin 2hao he 3hao ran se ti qiao di zu 。jie guo xian shi :dui zhao zu （wei qiao di qin ben zu ）guo ying zheng chang yuan luan ou bi li zai liang zhou nei dou gao da 92.0%yi shang ,biao ming luan chao GSCshu liang ji ben mei you bian hua 。yu hua hou 1d、7dhe 14d,laminji yin 2hao qiao di zu zheng chang yuan luan ou bi li fen bie wei 84.1%、36.7%he 31.2%;3hao qiao di zu zheng chang yuan luan ou de bi li fen bie wei 72.1%、54.8%he 36.6%。liang zu RNAishi yan jie guo xian shi :sui zhao shi jian tui yi ,nei yuan xing qiao di lamindao zhi zheng chang yuan luan ou de bi li ming xian jiang di ,an shi ji luan chao GSCshu liang fa sheng le yan chong de xia jiang 。dang wai yuan xing qiao di laminji yin ,shi yan zu tong ji jie guo yu dui zhao zu mo ming xian bian hua （P>0.05）,biao ming wai yuan xing qiao di laminbu ying xiang GSCwei chi 。ling wai ,cai yong you si fen lie chong zu ji shu nei yuan xing you dao GSCke long ,fen bie tong ji 1d、7dhe 14dbiao ji GSCke long de shu liang bian hua 。jie guo xian shi :ye sheng xing dui zhao zu biao ji de GSC（ji yin xing :FRT40A）de bai fen bi zai liang zhou nei cong 1dde 43.7%xia jiang dao 14dde 42.1%,GSCshu liang ji ben mo bian hua 。er san ge shi yan zu biao ji de lamintu bian (ji yin xing fen bie wei :FRT40A,lamA25、FRT40A,lamK2yu FRT40A,lam04643)de GSCbai fen bi cong di 1dde 43.8%、45.0%he 44.0%xia jiang dao di 14dde 15.1%、16.7%he 17.3%,liang zhou nei fen bie you 65.5%、62.9%he 60.7%de GSCfa sheng le diu shi ,zhi chi laminnei yuan xing de fa hui diao kong GSCwei chi de gong neng 。shang shu jie guo jun an shi laminji yin zai diao kong guo ying luan chao GSCwei chi fang mian fa hui nei yuan xing zuo yong 。wei le jin yi bu que zheng laminji yin de gong neng ,pai chu wei chuan bei jing de gan rao ,ben ke ti gou jian le yi ji lie laminzhuai ji yin zai ti ,fen bie cong nei yuan he wai yuan qu wan jiu laminji yin que shi dao zhi de biao xing 。jie guo xian shi :nei yuan xing bu chong laminji yin neng wan quan wan jiu laminque shi de biao xing ;jin wai yuan xing bu chong laminji yin mo fa wan jiu GSCde shu liang xia jiang ,biao ming laminji yin jin nei yuan xing diao kong guo ying luan chao GSCde wei chi 。ci wai ,jie zhu mian yi zu zhi hua xue ran se ji shu jian ce Lamindan bai de biao da mo shi ,guan cha fa xian ,Lamindan bai zai luan chao GSCszhong biao da liang gao ,ju ding wei yu xi bao he mo ,an shi le gai ji yin zai GSCzhong fa hui chong yao gong neng 。zong zhi ,shang shu jie guo chong fen biao ming laminji yin fa hui nei yuan xing diao kong guo ying luan chao GSCming yun de gong neng 。wei le que zheng laminji yin biao da yu GSCwei chi de xiang guan xing ,zai RNAshui ping shang ,li yong ying guang ding liang PCR（qPCR）jian ce le lamintu bian ti yu RNAgan rao pin ji guo ying luan chao zhong laminji yin de biao da 。jie guo xian shi ,yu ye sheng xing xiang bi ,tu bian ti he RNAgan rao zu zhong laminbiao da liang dou you ming xian jiang di 。zai dan bai zhi shui ping shang ,li yong dan bai mian yi yin ji （Western Blot）ji shu jian ce Lamindan bai de biao da 。jie guo xian shi :lamintu bian ti zhong Lamindan bai de biao da liang xian zhe jiang di 。ci jie guo chong fen biao ming laminji yin biao da xia diao shi dao zhi guo ying luan chao GSCsdiu shi de zhi jie yuan yin 。zai ci ,jie zhu bamP-GFPbao gao ji tong yan jiu laminyu yi zhi BMP/Dpp-bamxin hao tong lu de guan ji 。jie guo xian shi ,dui zhao zu zhong GFPyin xing biao ji de GSCshu zhan 97.7%,biao ming bamji yin chen mo shi GSCwei chi suo bi xu de 。zai lamintu bian de bei jing xia ,san zu shi yan zu guo ying (bamP-GFP;lamA25/lamA25、bamP-GFP;lamA25/lamK2he bamP-GFP;lamK2/lam04643)luan chao zhong ,fen bie you 18.8%、30.3%he 12.5%de GSCzhong jian ce dao GFPbiao ji de bamde biao da ,ming xian gao yu dui zhao zu 2.3%bi li 。gai jie guo biao ming laminji yin xiao bu fen de can yu le GSCzhong bamji yin de yi zhi （wei yu bamde shang you ）,tong shi an shi le laminji yin zhu yao wei yu bamde xia you ,huo tong guo ping hang yu BMP/Dpp-bamxin hao tong lu lai yi zhi GSCde fen hua yi wei chi gan xing 。ji ran lamintu bian dao zhi GSCshu liang diu shi ,na me que shi de GSCsshi fou zou xiang diao wang ?wei le hui da ci wen ti ,li yong TUNELji shu dui lamintu bian guo ying de luan chao GSCsjin hang diao wang jian ce 。jie guo xian shi ,ye sheng xing guo ying luan chao GSCdiao wang fa sheng lv wei 1.8%,liang chong lamintu bian ti luan chao GSCdiao wang fa sheng lv fen bie wei 5.1%he 5.3%,biao ming ye sheng xing yu tu bian ti guo ying de diao wang fa sheng lv mo ming xian cha yi 。ci wai ,li yong you si fen lie chong zu ji shu huo de de nei yuan xing lamintu bian GSCzhong ye mei you fa xian zeng jia de diao wang lv 。gai jie guo an shi le lamintu bian de GSCdi qian zou xiang fen hua er dao zhi diu shi 。wo men de gong zuo jia shen le dui GSCming yun jue ding ji zhi de li jie 。
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论文作者分别是来自安徽师范大学的孙福玲，发表于刊物安徽师范大学2019-07-10论文，是一篇关于基因论文,果蝇论文,卵巢生殖干细胞论文,命运论文,原卵区论文,安徽师范大学2019-07-10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，资料来自安徽师范大学2019-07-10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。