本文主要研究内容
作者孙平新（2019）在《人胚胎干细胞分化为自我更新的神经中胚层祖细胞研究》一文中研究指出：人胚胎干细胞(human embryonic stem cells,hESCs)在体外可以维持稳定自我更新,是能够分化产生所有类型成体细胞的多能干细胞(pluripotent stem cells,PSCs)。通过体外定向分化产生各种不同类型的细胞,可为细胞替代治疗提供潜在的细胞来源,在再生医学应用方面拥有巨大的发展前景。脊髓作为中枢神经系,具有上下传导神经信号、神经信号整合、协调运动等多种功能。肌萎缩性脊髓侧索硬化、神经性肌肉萎缩等脊髓退行性病变以及外源性创伤都将导致脊柱损伤,目前的手术、药物治疗方式对脊髓损伤的治疗效果有限,细胞移植有望成为脊髓损伤治疗的新手段。神经中胚层祖细胞(neuromesodermal progenitors,NMPs)不仅可以分化为脊髓中的神经细胞,而且还可以产生脊髓周边的中胚层组织,在脊髓损伤的细胞治疗中具有独特的应用价值。目前已经建立了较为成熟的诱导手段,将人PSCs分化为SOX2和T阳性的NMPs,但是如何实现NMPs的自我更新仍是有待解决的问题。本论文遵循体内发育线索,通过使用双重Smad抑制,同时激活WNT和FGF信号通路,成功获得了可以稳定自我更新的NMPs(self-renewing neuromesodermal progenitors,srNMPs)。该细胞共表达神经标志基因SOX2和中胚层标志基因T,同时还表达神经干细胞标志基因NESTIN、NCAD、CD133,增殖标志基因MKI67以及HOX家族基因HOXB8、HOXB9,验证了其脊髓后端神经干细胞的特征。srNMPs可在体外稳定传代超过30代,并维持细胞表型的稳定。在不添加任何形态发生素的神经分化条件下,srNMPs细胞分化产生vGLUT2、TACR1、SST、PAX2、HOXB8、HOXB9阳性的脊髓V0_G神经元。添加SHH、RA进行腹侧化诱导一周,srNMPs细胞被诱导为OLIG2阳性的运动神经前体细胞,神经分化三周后可产生共表达ISL1、ChAT、vAChT的运动神经元。激活WNT、BMP4进行背侧化诱导一周,再经过三周的神经分化,srNMPs细胞可分化为ATOH1、PAX3、PAX7阳性的脊髓dl1区中间神经元。该结果说明了srNMPs细胞可响应背腹轴信号的诱导,从而分化产生脊髓不同背腹轴定位的神经元。NMPs不仅是脊髓的前体细胞,而且是临近的轴旁中胚层(paraxial mesoderm)的前体细胞,轴旁中胚层主导了体节的形成,可进一步分化为软骨、骨骼肌、骨和真皮等中胚层组织。目前对NMPs的鉴定主要基于SOX2和T的共表达和向脊髓神经细胞的分化,NMPs在体外向中胚层细胞的分化尚有待验证。为了进一步证明srNMPs的中胚层分化潜能,我们通过单克隆分化、悬浮培养成球分化和体内肾包膜移植等实验来证明srNMPs具有神经和中胚层双向分化潜能。单克隆形成实验中,单个srNMPs细胞形成的克隆可在N2B27基础培养基中自发分化,可产生α-SMA阳性的中胚层细胞和MAP2阳性的神经元。同样,srNMPs也可在肾包膜下可分化成α-SMA阳性的中胚层组织和MAP2阳性的神经组织。悬浮培养实验中,srNMPs细胞成球后自发分化,可产生包含神经组织、软骨组织、肌肉组织的复杂结构,类似于胚胎发育过程中的脊柱。以上结果进一步证明了srNMPs细胞是神经中胚层祖细胞,具有中胚层和神经外胚层的双向分化潜能。本论文还对NMPs的发育起源及机制进行了一些探讨:1.使用小分子抑制剂SB431542、DMH1、LGK974分别抑制TGFβ、BMP、WNT信号通路,可将hESCs诱导成FOXG1阳性的前端神经上皮细胞,但是再激活WNT和FGF信号通路时,其无法转变为NMPs,从而证明了NMPs和前端神经外胚层细胞具有不同的发育起源。2.过表达FOXG1的细胞可以被成功诱导为NMPs,证明了前端神经外胚层细胞无法被诱导为NMPs的关键基因并非是FOXG1。3.有文献报道,SP8敲除小鼠的脊柱发育异常,所以本研究通过CRISPR/Cas9技术获得SP8敲除的hESCs,但是该细胞仍可被成功诱导为NMPs,证明了SP8不是NMPs诱导过程的关键基因。4.有报道证明,NMPs细胞中调控SOX2表达的增强子原件与ESCs、神经上皮细胞不同,NMPs细胞的SOX2的表达受增强子N1调控,ESCs和神经上皮细胞的SOX2的表达受增强子N2调控。通过甲基化测序检测,人的ESCs、NE及NMPs的SOX2增强子N1、N2中的甲基化状态,检测其是否存在差异。检测结果显示SOX2的增强子N1、N2的甲基化位点基本上没有发生甲基化现象,所以SOX2的增强子N1、N2的表达活性差异并非通过甲基化调控实现。综上,本研究成功获得了可以稳定自我更新的NMPs,该细胞共表达基因SOX2和T,可在体外稳定传代超过30代并维持细胞表型的稳定,具有脊髓神经干细胞的分子特征,能响应背腹轴信号分化产生脊髓背侧、腹侧神经元,并能在多种细胞分化实验中均能产生神经和中胚层细胞。悬浮培养的NMPs自发分化后形成包含神经组织、肌肉和软骨组织的细胞团,类似于发育中的脊柱。本课题建立的自我更新的NMPs有望在脊柱损伤的细胞治疗和NMPs细胞分化命运选择的机制研究中具有重要的应用价值。
Abstract
ren pei tai gan xi bao (human embryonic stem cells,hESCs)zai ti wai ke yi wei chi wen ding zi wo geng xin ,shi neng gou fen hua chan sheng suo you lei xing cheng ti xi bao de duo neng gan xi bao (pluripotent stem cells,PSCs)。tong guo ti wai ding xiang fen hua chan sheng ge chong bu tong lei xing de xi bao ,ke wei xi bao ti dai zhi liao di gong qian zai de xi bao lai yuan ,zai zai sheng yi xue ying yong fang mian yong you ju da de fa zhan qian jing 。ji sui zuo wei zhong shu shen jing ji ,ju you shang xia chuan dao shen jing xin hao 、shen jing xin hao zheng ge 、xie diao yun dong deng duo chong gong neng 。ji wei su xing ji sui ce suo ying hua 、shen jing xing ji rou wei su deng ji sui tui hang xing bing bian yi ji wai yuan xing chuang shang dou jiang dao zhi ji zhu sun shang ,mu qian de shou shu 、yao wu zhi liao fang shi dui ji sui sun shang de zhi liao xiao guo you xian ,xi bao yi zhi you wang cheng wei ji sui sun shang zhi liao de xin shou duan 。shen jing zhong pei ceng zu xi bao (neuromesodermal progenitors,NMPs)bu jin ke yi fen hua wei ji sui zhong de shen jing xi bao ,er ju hai ke yi chan sheng ji sui zhou bian de zhong pei ceng zu zhi ,zai ji sui sun shang de xi bao zhi liao zhong ju you du te de ying yong jia zhi 。mu qian yi jing jian li le jiao wei cheng shou de you dao shou duan ,jiang ren PSCsfen hua wei SOX2he Tyang xing de NMPs,dan shi ru he shi xian NMPsde zi wo geng xin reng shi you dai jie jue de wen ti 。ben lun wen zun xun ti nei fa yo xian suo ,tong guo shi yong shuang chong Smadyi zhi ,tong shi ji huo WNThe FGFxin hao tong lu ,cheng gong huo de le ke yi wen ding zi wo geng xin de NMPs(self-renewing neuromesodermal progenitors,srNMPs)。gai xi bao gong biao da shen jing biao zhi ji yin SOX2he zhong pei ceng biao zhi ji yin T,tong shi hai biao da shen jing gan xi bao biao zhi ji yin NESTIN、NCAD、CD133,zeng shi biao zhi ji yin MKI67yi ji HOXjia zu ji yin HOXB8、HOXB9,yan zheng le ji ji sui hou duan shen jing gan xi bao de te zheng 。srNMPske zai ti wai wen ding chuan dai chao guo 30dai ,bing wei chi xi bao biao xing de wen ding 。zai bu tian jia ren he xing tai fa sheng su de shen jing fen hua tiao jian xia ,srNMPsxi bao fen hua chan sheng vGLUT2、TACR1、SST、PAX2、HOXB8、HOXB9yang xing de ji sui V0_Gshen jing yuan 。tian jia SHH、RAjin hang fu ce hua you dao yi zhou ,srNMPsxi bao bei you dao wei OLIG2yang xing de yun dong shen jing qian ti xi bao ,shen jing fen hua san zhou hou ke chan sheng gong biao da ISL1、ChAT、vAChTde yun dong shen jing yuan 。ji huo WNT、BMP4jin hang bei ce hua you dao yi zhou ,zai jing guo san zhou de shen jing fen hua ,srNMPsxi bao ke fen hua wei ATOH1、PAX3、PAX7yang xing de ji sui dl1ou zhong jian shen jing yuan 。gai jie guo shui ming le srNMPsxi bao ke xiang ying bei fu zhou xin hao de you dao ,cong er fen hua chan sheng ji sui bu tong bei fu zhou ding wei de shen jing yuan 。NMPsbu jin shi ji sui de qian ti xi bao ,er ju shi lin jin de zhou pang zhong pei ceng (paraxial mesoderm)de qian ti xi bao ,zhou pang zhong pei ceng zhu dao le ti jie de xing cheng ,ke jin yi bu fen hua wei ruan gu 、gu ge ji 、gu he zhen pi deng zhong pei ceng zu zhi 。mu qian dui NMPsde jian ding zhu yao ji yu SOX2he Tde gong biao da he xiang ji sui shen jing xi bao de fen hua ,NMPszai ti wai xiang zhong pei ceng xi bao de fen hua shang you dai yan zheng 。wei le jin yi bu zheng ming srNMPsde zhong pei ceng fen hua qian neng ,wo men tong guo chan ke long fen hua 、xuan fu pei yang cheng qiu fen hua he ti nei shen bao mo yi zhi deng shi yan lai zheng ming srNMPsju you shen jing he zhong pei ceng shuang xiang fen hua qian neng 。chan ke long xing cheng shi yan zhong ,chan ge srNMPsxi bao xing cheng de ke long ke zai N2B27ji chu pei yang ji zhong zi fa fen hua ,ke chan sheng α-SMAyang xing de zhong pei ceng xi bao he MAP2yang xing de shen jing yuan 。tong yang ,srNMPsye ke zai shen bao mo xia ke fen hua cheng α-SMAyang xing de zhong pei ceng zu zhi he MAP2yang xing de shen jing zu zhi 。xuan fu pei yang shi yan zhong ,srNMPsxi bao cheng qiu hou zi fa fen hua ,ke chan sheng bao han shen jing zu zhi 、ruan gu zu zhi 、ji rou zu zhi de fu za jie gou ,lei shi yu pei tai fa yo guo cheng zhong de ji zhu 。yi shang jie guo jin yi bu zheng ming le srNMPsxi bao shi shen jing zhong pei ceng zu xi bao ,ju you zhong pei ceng he shen jing wai pei ceng de shuang xiang fen hua qian neng 。ben lun wen hai dui NMPsde fa yo qi yuan ji ji zhi jin hang le yi xie tan tao :1.shi yong xiao fen zi yi zhi ji SB431542、DMH1、LGK974fen bie yi zhi TGFβ、BMP、WNTxin hao tong lu ,ke jiang hESCsyou dao cheng FOXG1yang xing de qian duan shen jing shang pi xi bao ,dan shi zai ji huo WNThe FGFxin hao tong lu shi ,ji mo fa zhuai bian wei NMPs,cong er zheng ming le NMPshe qian duan shen jing wai pei ceng xi bao ju you bu tong de fa yo qi yuan 。2.guo biao da FOXG1de xi bao ke yi bei cheng gong you dao wei NMPs,zheng ming le qian duan shen jing wai pei ceng xi bao mo fa bei you dao wei NMPsde guan jian ji yin bing fei shi FOXG1。3.you wen suo bao dao ,SP8qiao chu xiao shu de ji zhu fa yo yi chang ,suo yi ben yan jiu tong guo CRISPR/Cas9ji shu huo de SP8qiao chu de hESCs,dan shi gai xi bao reng ke bei cheng gong you dao wei NMPs,zheng ming le SP8bu shi NMPsyou dao guo cheng de guan jian ji yin 。4.you bao dao zheng ming ,NMPsxi bao zhong diao kong SOX2biao da de zeng jiang zi yuan jian yu ESCs、shen jing shang pi xi bao bu tong ,NMPsxi bao de SOX2de biao da shou zeng jiang zi N1diao kong ,ESCshe shen jing shang pi xi bao de SOX2de biao da shou zeng jiang zi N2diao kong 。tong guo jia ji hua ce xu jian ce ,ren de ESCs、NEji NMPsde SOX2zeng jiang zi N1、N2zhong de jia ji hua zhuang tai ,jian ce ji shi fou cun zai cha yi 。jian ce jie guo xian shi SOX2de zeng jiang zi N1、N2de jia ji hua wei dian ji ben shang mei you fa sheng jia ji hua xian xiang ,suo yi SOX2de zeng jiang zi N1、N2de biao da huo xing cha yi bing fei tong guo jia ji hua diao kong shi xian 。zeng shang ,ben yan jiu cheng gong huo de le ke yi wen ding zi wo geng xin de NMPs,gai xi bao gong biao da ji yin SOX2he T,ke zai ti wai wen ding chuan dai chao guo 30dai bing wei chi xi bao biao xing de wen ding ,ju you ji sui shen jing gan xi bao de fen zi te zheng ,neng xiang ying bei fu zhou xin hao fen hua chan sheng ji sui bei ce 、fu ce shen jing yuan ,bing neng zai duo chong xi bao fen hua shi yan zhong jun neng chan sheng shen jing he zhong pei ceng xi bao 。xuan fu pei yang de NMPszi fa fen hua hou xing cheng bao han shen jing zu zhi 、ji rou he ruan gu zu zhi de xi bao tuan ,lei shi yu fa yo zhong de ji zhu 。ben ke ti jian li de zi wo geng xin de NMPsyou wang zai ji zhu sun shang de xi bao zhi liao he NMPsxi bao fen hua ming yun shua ze de ji zhi yan jiu zhong ju you chong yao de ying yong jia zhi 。
论文参考文献
论文详细介绍
论文作者分别是来自中国人民解放军海军军医大学的孙平新，发表于刊物中国人民解放军海军军医大学2019-07-01论文，是一篇关于人胚胎干细胞论文,多能干细胞论文,神经中胚层祖细胞论文,自我更新论文,后端神经板论文,细胞治疗论文,中国人民解放军海军军医大学2019-07-01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，资料来自中国人民解放军海军军医大学2019-07-01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：人胚胎干细胞论文; 多能干细胞论文; 神经中胚层祖细胞论文; 自我更新论文; 后端神经板论文; 细胞治疗论文; 中国人民解放军海军军医大学2019-07-01论文;