本文主要研究内容
作者钟亚东（2019）在《基于Cre/loxP改进增强子捕获研究肝脏和胰腺β细胞的发育和再生》一文中研究指出：肝脏和胰腺是来源于胚胎内胚层的两个重要的消化器官。肝脏在新陈代谢过程中发挥着广泛的作用,包括糖类代谢,脂类的体内平衡和酮体的产生,氨基酸的新陈代谢,对摄入各类化合物的解毒以及维持血液中代谢物和蛋白质的生理浓度。肝实质细胞(占肝脏细胞类型70-75%)负责执行绝大多数这些肝功能,并且和胆管上皮细胞(占肝脏细胞类型10-15%)一起组成肝实质。胰腺分别由执行内分泌功能和外分泌功能的两类腺体构成。外分泌腺细胞产生并且分泌消化酶,占整个胰腺细胞质量的95%。四种胰腺内分泌细胞负责产生不同的胰腺激素,α细胞产生胰高血糖素,β细胞产生胰岛素,δ细胞产生抑生长素,PP细胞产生胰腺多肽。斑马鱼和哺乳动物的肝脏和胰腺在发育的分子调控方面显示出惊人的相似性,它们拥有相同的细胞和亚细胞结构,并且共同拥有保守的生理学功能。所有这些标准都使得斑马鱼能作为一种研究肝脏和胰腺β细胞发育和再生的可靠动物模型。成体中的肝胰器官都具有维持组织更新和损伤后再生的能力,既可以是通过剩余细胞复制而来,也可以通过多潜能的前体细胞分化而来,或则由其他类型的细胞转分化而来。哺乳动物的研究提供了以上所有机制都存在的证据,具体由哪种机制参与肝胰器官的稳态维持和损伤后的再生应答取决于具体的实验操作和分析方法。但是,具体有哪些前体细胞或分化的细胞能贡献给肝胰组织的稳态平衡或者损伤后的再生,以及控制这个过程的分子途径仍然充满了未知。因此去寻找参与肝脏和胰腺β细胞维持稳态或者参与损伤后再生的前体细胞,以及控制这一过程的关键调控基因将是非常引人入胜的研究。目前还未见到肝脏和胰腺β细胞在正常发育和损伤后再生过程中,能直观地在活体中反映特异基因表达的方法手段。在本研究中,我们以组织特异性标记肝脏和胰腺β细胞的转基因斑马鱼为动物模型,结合增强子捕获,大规模地筛选参与肝胰发育,维持生理功能和损伤后再生的相关基因。我们分别构建了标记肝实质和胰腺β细胞的转基因斑马鱼,Tg(fabp10:loxPCFPNTR-loxP-DsRed)和Tg(ins:loxP-CFPNTR-loxP-DsRed),然后再与Tg(10×UAS:Cre,cryaa:Venus)杂交,分别得到以Cre/loxp系统为基础的标记肝脏和胰腺的双转基因鱼Tg(fabp10:loxP-CFPNTR-loxP-DsRed;10×UAS:Cre,cryaa:Venus)和Tg(ins:loxP-CFPNTR-loxP-DsRed;10×UAS:Cre,cryaa:Venus)。与CFP荧光蛋白所融合的硝基还原酶NTR能把无害的剧毒前体MtZ还原为剧毒,从而导致所含NTR的细胞的死亡。我们发现在10毫摩尔浓度MtZ处理24小时后,肝实质细胞和胰腺β细胞都能被完全地诱导凋亡,并且在撤除药物后的24小时和48小时出现明显的再生。通过大量注射含有转座子原件Tol2的GAL4到1细胞期胚胎,我们构建了1000条增强子捕获的F0.在肝脏和胰腺的转基因鱼Tg(fabp10:loxPCFPNTR-loxP-DsRed;10×UAS:Cre,cryaa:Venus)和Tg(ins:loxP-CFPNTR-loxPDsRed;10×UAS:Cre,cryaa:Venus)中,含有在上游激活元件UAS控制下表达的同源重组酶CRE,只有当UAS元件被GAL4转录因子结合的情况下,Cre才能在细胞质表达。让这些增强子捕获F0分别与标记肝脏和胰腺的Tg(fabp10:loxPCFPNTR-loxP-DsRed;10×UAS:Cre,cryaa:Venus)和Tg(ins:loxP-CFPNTR-loxPDsRed;10×UAS:Cre,cryaa:Venus)杂交,然后观察肝脏和胰腺β细胞的荧光标记的颜色变化,从而筛选潜在的肝胰特异性插入。通过1000次杂交筛选,我们得到了一些能使肝脏和胰腺β细胞从蓝色标记变成红色标记的增强子插入,然后定位插入的基因组位置。所有靠近插入位置的候选基因都用原为杂交鉴定能否在肝胰特异性表达的基因。同时运用转基因品系的优势,用能在硝基还原酶Nitroreductase作用下变成细胞毒素的甲硝唑Mtz,特异性的损伤了肝脏实质细胞和胰腺β细胞,并且对这两类细胞的再生过程进行了筛选。我们运用此种方法在肝脏和胰腺β细胞发育和再生过程中,都鉴定出了组织特异性表达的基因,并且对此永久谱系标记方法与传统增强子捕获的效果进行了比较,我们发现基于Cre/LoxP的增强子捕获能筛选到发育再生过程中瞬间表达和相对较弱表达的基因。值得注意的是,在肝脏发育过程中表达的基因rnd2,在β细胞发育过程中表达的基因rab3da和在肝脏再生过程中表达的基因ensab在此改进的筛选中表现出比传统增强子捕获更高的效率。因此,运用永久标记的增强子捕获为筛选器官发育和再生过程中较弱表达,瞬时表达又潜在重要的基因提供了有运用前景的方法。
Abstract
gan zang he yi xian shi lai yuan yu pei tai nei pei ceng de liang ge chong yao de xiao hua qi guan 。gan zang zai xin chen dai xie guo cheng zhong fa hui zhao an fan de zuo yong ,bao gua tang lei dai xie ,zhi lei de ti nei ping heng he tong ti de chan sheng ,an ji suan de xin chen dai xie ,dui she ru ge lei hua ge wu de jie du yi ji wei chi xie ye zhong dai xie wu he dan bai zhi de sheng li nong du 。gan shi zhi xi bao (zhan gan zang xi bao lei xing 70-75%)fu ze zhi hang jue da duo shu zhe xie gan gong neng ,bing ju he dan guan shang pi xi bao (zhan gan zang xi bao lei xing 10-15%)yi qi zu cheng gan shi zhi 。yi xian fen bie you zhi hang nei fen bi gong neng he wai fen bi gong neng de liang lei xian ti gou cheng 。wai fen bi xian xi bao chan sheng bing ju fen bi xiao hua mei ,zhan zheng ge yi xian xi bao zhi liang de 95%。si chong yi xian nei fen bi xi bao fu ze chan sheng bu tong de yi xian ji su ,αxi bao chan sheng yi gao xie tang su ,βxi bao chan sheng yi dao su ,δxi bao chan sheng yi sheng chang su ,PPxi bao chan sheng yi xian duo tai 。ban ma yu he bu ru dong wu de gan zang he yi xian zai fa yo de fen zi diao kong fang mian xian shi chu jing ren de xiang shi xing ,ta men yong you xiang tong de xi bao he ya xi bao jie gou ,bing ju gong tong yong you bao shou de sheng li xue gong neng 。suo you zhe xie biao zhun dou shi de ban ma yu neng zuo wei yi chong yan jiu gan zang he yi xian βxi bao fa yo he zai sheng de ke kao dong wu mo xing 。cheng ti zhong de gan yi qi guan dou ju you wei chi zu zhi geng xin he sun shang hou zai sheng de neng li ,ji ke yi shi tong guo sheng yu xi bao fu zhi er lai ,ye ke yi tong guo duo qian neng de qian ti xi bao fen hua er lai ,huo ze you ji ta lei xing de xi bao zhuai fen hua er lai 。bu ru dong wu de yan jiu di gong le yi shang suo you ji zhi dou cun zai de zheng ju ,ju ti you na chong ji zhi can yu gan yi qi guan de wen tai wei chi he sun shang hou de zai sheng ying da qu jue yu ju ti de shi yan cao zuo he fen xi fang fa 。dan shi ,ju ti you na xie qian ti xi bao huo fen hua de xi bao neng gong suo gei gan yi zu zhi de wen tai ping heng huo zhe sun shang hou de zai sheng ,yi ji kong zhi zhe ge guo cheng de fen zi tu jing reng ran chong man le wei zhi 。yin ci qu xun zhao can yu gan zang he yi xian βxi bao wei chi wen tai huo zhe can yu sun shang hou zai sheng de qian ti xi bao ,yi ji kong zhi zhe yi guo cheng de guan jian diao kong ji yin jiang shi fei chang yin ren ru sheng de yan jiu 。mu qian hai wei jian dao gan zang he yi xian βxi bao zai zheng chang fa yo he sun shang hou zai sheng guo cheng zhong ,neng zhi guan de zai huo ti zhong fan ying te yi ji yin biao da de fang fa shou duan 。zai ben yan jiu zhong ,wo men yi zu zhi te yi xing biao ji gan zang he yi xian βxi bao de zhuai ji yin ban ma yu wei dong wu mo xing ,jie ge zeng jiang zi bu huo ,da gui mo de shai shua can yu gan yi fa yo ,wei chi sheng li gong neng he sun shang hou zai sheng de xiang guan ji yin 。wo men fen bie gou jian le biao ji gan shi zhi he yi xian βxi bao de zhuai ji yin ban ma yu ,Tg(fabp10:loxPCFPNTR-loxP-DsRed)he Tg(ins:loxP-CFPNTR-loxP-DsRed),ran hou zai yu Tg(10×UAS:Cre,cryaa:Venus)za jiao ,fen bie de dao yi Cre/loxpji tong wei ji chu de biao ji gan zang he yi xian de shuang zhuai ji yin yu Tg(fabp10:loxP-CFPNTR-loxP-DsRed;10×UAS:Cre,cryaa:Venus)he Tg(ins:loxP-CFPNTR-loxP-DsRed;10×UAS:Cre,cryaa:Venus)。yu CFPying guang dan bai suo rong ge de xiao ji hai yuan mei NTRneng ba mo hai de ju du qian ti MtZhai yuan wei ju du ,cong er dao zhi suo han NTRde xi bao de si wang 。wo men fa xian zai 10hao ma er nong du MtZchu li 24xiao shi hou ,gan shi zhi xi bao he yi xian βxi bao dou neng bei wan quan de you dao diao wang ,bing ju zai che chu yao wu hou de 24xiao shi he 48xiao shi chu xian ming xian de zai sheng 。tong guo da liang zhu she han you zhuai zuo zi yuan jian Tol2de GAL4dao 1xi bao ji pei tai ,wo men gou jian le 1000tiao zeng jiang zi bu huo de F0.zai gan zang he yi xian de zhuai ji yin yu Tg(fabp10:loxPCFPNTR-loxP-DsRed;10×UAS:Cre,cryaa:Venus)he Tg(ins:loxP-CFPNTR-loxPDsRed;10×UAS:Cre,cryaa:Venus)zhong ,han you zai shang you ji huo yuan jian UASkong zhi xia biao da de tong yuan chong zu mei CRE,zhi you dang UASyuan jian bei GAL4zhuai lu yin zi jie ge de qing kuang xia ,Crecai neng zai xi bao zhi biao da 。rang zhe xie zeng jiang zi bu huo F0fen bie yu biao ji gan zang he yi xian de Tg(fabp10:loxPCFPNTR-loxP-DsRed;10×UAS:Cre,cryaa:Venus)he Tg(ins:loxP-CFPNTR-loxPDsRed;10×UAS:Cre,cryaa:Venus)za jiao ,ran hou guan cha gan zang he yi xian βxi bao de ying guang biao ji de yan se bian hua ,cong er shai shua qian zai de gan yi te yi xing cha ru 。tong guo 1000ci za jiao shai shua ,wo men de dao le yi xie neng shi gan zang he yi xian βxi bao cong lan se biao ji bian cheng gong se biao ji de zeng jiang zi cha ru ,ran hou ding wei cha ru de ji yin zu wei zhi 。suo you kao jin cha ru wei zhi de hou shua ji yin dou yong yuan wei za jiao jian ding neng fou zai gan yi te yi xing biao da de ji yin 。tong shi yun yong zhuai ji yin pin ji de you shi ,yong neng zai xiao ji hai yuan mei Nitroreductasezuo yong xia bian cheng xi bao du su de jia xiao zuo Mtz,te yi xing de sun shang le gan zang shi zhi xi bao he yi xian βxi bao ,bing ju dui zhe liang lei xi bao de zai sheng guo cheng jin hang le shai shua 。wo men yun yong ci chong fang fa zai gan zang he yi xian βxi bao fa yo he zai sheng guo cheng zhong ,dou jian ding chu le zu zhi te yi xing biao da de ji yin ,bing ju dui ci yong jiu pu ji biao ji fang fa yu chuan tong zeng jiang zi bu huo de xiao guo jin hang le bi jiao ,wo men fa xian ji yu Cre/LoxPde zeng jiang zi bu huo neng shai shua dao fa yo zai sheng guo cheng zhong shun jian biao da he xiang dui jiao ruo biao da de ji yin 。zhi de zhu yi de shi ,zai gan zang fa yo guo cheng zhong biao da de ji yin rnd2,zai βxi bao fa yo guo cheng zhong biao da de ji yin rab3dahe zai gan zang zai sheng guo cheng zhong biao da de ji yin ensabzai ci gai jin de shai shua zhong biao xian chu bi chuan tong zeng jiang zi bu huo geng gao de xiao lv 。yin ci ,yun yong yong jiu biao ji de zeng jiang zi bu huo wei shai shua qi guan fa yo he zai sheng guo cheng zhong jiao ruo biao da ,shun shi biao da you qian zai chong yao de ji yin di gong le you yun yong qian jing de fang fa 。
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论文作者分别是来自西南大学的钟亚东，发表于刊物西南大学2019-09-24论文，是一篇关于增强子捕获论文,肝脏论文,胰腺细胞论文,发育论文,再生论文,西南大学2019-09-24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，资料来自西南大学2019-09-24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。