本文主要研究内容
作者展延栋（2019）在《斑马鱼尾动脉经内皮造血转化产生造血干细胞及前体细胞》一文中研究指出：造血干细胞(HSC)是血液系统各细胞组分的共同祖细胞,它通过不断增殖和有序分化,形成了整个血液组分,同时通过持续的自我更新维持着造血干细胞和整个血液谱系的稳态。造血干细胞形成、维持、增殖以及分化的缺陷会导致多种血液系统及免疫系统的疾病,如白血病、贫血、类风湿和红斑狼疮等。因此,造血干细胞的正常形成对于机体的健康起着至关重要的作用。与此同时,治疗血液系统疾病的一个重要方法是造血干细胞移植,对于造血干细胞的形成和调控机制需深入认知,体外诱导造血干细胞的生成及扩增是实现移植治疗的先决条件。所以,关于造血干细胞的相关研究,尤其是造血干细胞的起源过程、谱系分化和细胞形成的分子调控机制的研究一直以来都是生命科学和医学领域的热点。通过对小鼠等模式动物进行深入的研究,研究者发现原始造血发生在卵黄囊,这些血液细胞短暂存在并主要分化为原始红系和髓系祖细胞。随着胚胎的发育定向造血出现在AGM中,因为最早检测到造血干细胞的出现而被认为是造血干细胞产生的位置,其中一部分位于主动脉腹侧壁的血管内皮细胞会特化为生血内皮细胞并在特定的胚胎发育时间窗,通过内皮-造血转化过程(EHT)分化为造血干细胞。近年来通过检测造血干细胞的标记分子,谱系追踪和移植等一系列实验,科研工作者发现胚胎期小鼠的脑血管也能够原位产生造血干细胞,且这群造血干细胞的发生几乎与AGM来源同步。在胚胎阶段除了AGM区的生血内皮细胞外,其他部位的胚胎细胞能够重建成体小鼠的造血系统,这促使科研工作者怀疑在胚胎发育的特定时间段是否还存在着其他的原位产生造血干细胞的器官或组织。在对造血干细胞的发生过程和调控机制的认知中,斑马鱼扮演了不可替代的重要角色。通过对斑马鱼活体实时成像研究者直接证实了造血干细胞通过内皮-造血转化过程的方式源于背侧主动脉(DA)腹侧壁的生血内皮细胞,后会进一步迁移到后部血岛组织(caudal hematopoietic tissue,CHT)进行增殖和分化,最终这些造血干细胞会迁移到胸腺和肾脏(与高等动物的骨髓功能类似),为整个斑马鱼一生血液系统的维持提供着源源不断的支持。CHT的功能和小鼠的胎肝相似,造血干细胞迁移到CHT后与微环境的各组成细胞之间相互作用的动态过程,是造血干细胞进一步增殖和分化的区域。但是CHT是否可以原位产生造血干细胞是从没有被研究的全新课题。本课题首先通过活体实时成像,发现在斑马鱼CHT的尾部主动脉(caudal dorsal artery,CDA)腹侧壁一些kdrl-EGFP+细胞会出现DA区类似的EHT过程,这些细胞最终会被标记HSPCs的CD41-GFP、cmyb-GFP和runx1-GFP所标记,偶尔可以观察到kdrl-EGFP+细胞出芽后会分裂。我们利用共聚焦405激发波长对转基因品系Tg(kdrl:Dendra2)进行光转化,标记部分尾部动脉腹侧壁,之后可看到造血区域如CHT,胸腺和肾脏都存在被标记的细胞。对12个胚胎斑马鱼CDA进行活体实时成像观察,其中大约在DA的EHT过程之后60 hpf(hours post fertilization)左右,CDA与尾部静脉分开,CHT的EHT过程开始,一直持续到156 hpf。CDA大部分的EHT过程发生在60 hpf-84 hpf,随时间的推移,EHT频率慢慢下降。不同时间段60-84,84-108,108-132,132-156和156-180 hpf EHT的频率(不同时间段四个体节的出芽频率)分别是5.60±0.68,3.40±0.81,1.50±0.40,0.80±0.58和0.0±0.0。在统计的15小时中,每四个体节CDA出芽频率(3.57±0.57)低于DA出芽频率(5.86±0.26)。因此,CDA在时空上独立于DA,通过EHT过程产生HSPCs。为了进一步探索CDA产生的HSPCs,我们利用Tg(CD41:GFP;coro1a:Ds Red),Tg(kdrl:mem Cherry;coro1a:Kaede)追踪DA和CDA刚产生的HSPCs的行为特征。刚产生的细胞中含有coro1a-Ds Red信号和CD41-GFP弱信号,并且coro1a-Kaede信号也在刚出芽的kdrl-mem Cherry+细胞中表达,这说明我们可以用coro1a-Kaede光转化来标记DA和CDA刚产生的HSPCs。结果显示,CDA出芽产生的coro1a-Kaede细胞大约30%-40%很快分裂成两个子细胞,12小时内会进行第二次分裂,随后一些细胞会进入血管和血液循环,另一部分细胞会保持原位置进行第三次分裂。其中,偶尔能观察到一些细胞进入血液循环后会再次进入CHT微环境。CDA产生的细胞在细胞行为学上很像HSPCs的行为,形态上成球形分裂成前体细胞,体积分裂后会变小。相比之下,CDA产生的coro1a-Kaede+细胞一大半经过一次分裂会产生髓样的前体细胞,细胞形态呈变形虫形态而且变大。经过10个小时,这些髓样前体细胞会分裂成3-4个子细胞,迁移到CHT微环境中呈现分支装表型,一些细胞会随血管迁出CHT并迁移至皮肤,呈现典型的髓系细胞的行为。大约有2%-5%被标记的细胞在出芽时会破碎死掉。通过光转化标记DA和CDA出芽的coro1a-Kaede+细胞,经过60小时的追踪,这些细胞行为学的追踪表明CDA产生的HSPCs存在着异质性。由于DA和CDA产生的HSPCs在时空上和迁移倾向上有所不同,我们探讨其中潜在的调控分子。Runx1对于DA的EHT过程来说是必不可少的转录因子,那Runx1是如何影响CDA区的EHT过程的呢?我们利用斑马鱼突变体runx1w84x/w84xTg(kdrl:EGFP),发现CDA无法完成EHT过程,有些细胞会有出芽的趋势,但是最后都收缩回去。这说明CDA和DA一样,EHT过程都需要转录因此Runx1来调控。在造血过程中,血流对血管剪切应力促进了造血干细胞的产生。我们利用无血流的tnnt2aswu77斑马鱼突变体,发现CDA的EHT过程被抑制,而且大部分HSPCs向CDA血管内侧出芽,而DA向腹侧出芽产生的HSPCs。上述结果表明,从DA到CDA连续发生的EHT过程,为整个动脉都有造血能力提供了证据。为了进一步探究动脉不同时间段对成体斑马鱼造血的贡献情况,我们利用Tg(kdrl:Cre ERT2;actb2:lox P-Ds Red-stop-lox P-EGFP)s928转基因系统,在五个时间点20 hpf,60 hpf,72 hpf,120 hpf和9 dpf(days post fertilization)对斑马鱼胚胎进行4-OHT(4-Hydroxytamoxifen)处理,并检测出4-OHT只诱导血管及血液细胞Cre重组酶活性。对4个月零后的成年斑马鱼的不同组织区域进行切片免疫荧光染色。利用GFP和Lcp1两个抗体共同标记出早期胚胎来源的造血细胞,20 hpf处理组中,成年斑马鱼造血组织中GFP阳性细胞占Lcp1+细胞的比例最高,肾脏中占比76%,胸腺中占比89%,随着处理时间的推移,GFP+Lcp1+细胞比例下降。相比于20 hpf处理组,60 hpf处理组中GFP+Lcp1+细胞在胸腺比例下降的幅度要比肾脏中的轻微一些,这暗示着这一时段动脉造血对成年胸腺淋巴细胞贡献较大。与此同时,大多数成年斑马鱼中脑部小胶质细胞由kdrl+细胞分化而来,这支持了定向造血来源地小胶质细胞的观点。之后,我们在Tg(kdrl:Cre ERT2;coro1a:lox P-Ds Redx-stop-lox P-GFP)s928转基因系统里用了相同处理方法,得出了上述相似的结论。因此,随着时间的推移,kdrl+动脉内皮细胞通过EHT过程对成年造血谱系的贡献能力逐渐减弱。根据我们的研究表明,在斑马鱼中位于CHT区的CDA作为新发现的造血位点,与AGM区的DA类似,具有通过EHT过程产生HSPCs的能力,并且产生的HSPCs迁移至不同造血组织器官。我们的发现扩展了斑马鱼造血发育过程的理论研究。
Abstract
zao xie gan xi bao (HSC)shi xie ye ji tong ge xi bao zu fen de gong tong zu xi bao ,ta tong guo bu duan zeng shi he you xu fen hua ,xing cheng le zheng ge xie ye zu fen ,tong shi tong guo chi xu de zi wo geng xin wei chi zhao zao xie gan xi bao he zheng ge xie ye pu ji de wen tai 。zao xie gan xi bao xing cheng 、wei chi 、zeng shi yi ji fen hua de que xian hui dao zhi duo chong xie ye ji tong ji mian yi ji tong de ji bing ,ru bai xie bing 、pin xie 、lei feng shi he gong ban lang chuang deng 。yin ci ,zao xie gan xi bao de zheng chang xing cheng dui yu ji ti de jian kang qi zhao zhi guan chong yao de zuo yong 。yu ci tong shi ,zhi liao xie ye ji tong ji bing de yi ge chong yao fang fa shi zao xie gan xi bao yi zhi ,dui yu zao xie gan xi bao de xing cheng he diao kong ji zhi xu shen ru ren zhi ,ti wai you dao zao xie gan xi bao de sheng cheng ji kuo zeng shi shi xian yi zhi zhi liao de xian jue tiao jian 。suo yi ,guan yu zao xie gan xi bao de xiang guan yan jiu ,you ji shi zao xie gan xi bao de qi yuan guo cheng 、pu ji fen hua he xi bao xing cheng de fen zi diao kong ji zhi de yan jiu yi zhi yi lai dou shi sheng ming ke xue he yi xue ling yu de re dian 。tong guo dui xiao shu deng mo shi dong wu jin hang shen ru de yan jiu ,yan jiu zhe fa xian yuan shi zao xie fa sheng zai luan huang nang ,zhe xie xie ye xi bao duan zan cun zai bing zhu yao fen hua wei yuan shi gong ji he sui ji zu xi bao 。sui zhao pei tai de fa yo ding xiang zao xie chu xian zai AGMzhong ,yin wei zui zao jian ce dao zao xie gan xi bao de chu xian er bei ren wei shi zao xie gan xi bao chan sheng de wei zhi ,ji zhong yi bu fen wei yu zhu dong mai fu ce bi de xie guan nei pi xi bao hui te hua wei sheng xie nei pi xi bao bing zai te ding de pei tai fa yo shi jian chuang ,tong guo nei pi -zao xie zhuai hua guo cheng (EHT)fen hua wei zao xie gan xi bao 。jin nian lai tong guo jian ce zao xie gan xi bao de biao ji fen zi ,pu ji zhui zong he yi zhi deng yi ji lie shi yan ,ke yan gong zuo zhe fa xian pei tai ji xiao shu de nao xie guan ye neng gou yuan wei chan sheng zao xie gan xi bao ,ju zhe qun zao xie gan xi bao de fa sheng ji hu yu AGMlai yuan tong bu 。zai pei tai jie duan chu le AGMou de sheng xie nei pi xi bao wai ,ji ta bu wei de pei tai xi bao neng gou chong jian cheng ti xiao shu de zao xie ji tong ,zhe cu shi ke yan gong zuo zhe huai yi zai pei tai fa yo de te ding shi jian duan shi fou hai cun zai zhao ji ta de yuan wei chan sheng zao xie gan xi bao de qi guan huo zu zhi 。zai dui zao xie gan xi bao de fa sheng guo cheng he diao kong ji zhi de ren zhi zhong ,ban ma yu ban yan le bu ke ti dai de chong yao jiao se 。tong guo dui ban ma yu huo ti shi shi cheng xiang yan jiu zhe zhi jie zheng shi le zao xie gan xi bao tong guo nei pi -zao xie zhuai hua guo cheng de fang shi yuan yu bei ce zhu dong mai (DA)fu ce bi de sheng xie nei pi xi bao ,hou hui jin yi bu qian yi dao hou bu xie dao zu zhi (caudal hematopoietic tissue,CHT)jin hang zeng shi he fen hua ,zui zhong zhe xie zao xie gan xi bao hui qian yi dao xiong xian he shen zang (yu gao deng dong wu de gu sui gong neng lei shi ),wei zheng ge ban ma yu yi sheng xie ye ji tong de wei chi di gong zhao yuan yuan bu duan de zhi chi 。CHTde gong neng he xiao shu de tai gan xiang shi ,zao xie gan xi bao qian yi dao CHThou yu wei huan jing de ge zu cheng xi bao zhi jian xiang hu zuo yong de dong tai guo cheng ,shi zao xie gan xi bao jin yi bu zeng shi he fen hua de ou yu 。dan shi CHTshi fou ke yi yuan wei chan sheng zao xie gan xi bao shi cong mei you bei yan jiu de quan xin ke ti 。ben ke ti shou xian tong guo huo ti shi shi cheng xiang ,fa xian zai ban ma yu CHTde wei bu zhu dong mai (caudal dorsal artery,CDA)fu ce bi yi xie kdrl-EGFP+xi bao hui chu xian DAou lei shi de EHTguo cheng ,zhe xie xi bao zui zhong hui bei biao ji HSPCsde CD41-GFP、cmyb-GFPhe runx1-GFPsuo biao ji ,ou er ke yi guan cha dao kdrl-EGFP+xi bao chu ya hou hui fen lie 。wo men li yong gong ju jiao 405ji fa bo chang dui zhuai ji yin pin ji Tg(kdrl:Dendra2)jin hang guang zhuai hua ,biao ji bu fen wei bu dong mai fu ce bi ,zhi hou ke kan dao zao xie ou yu ru CHT,xiong xian he shen zang dou cun zai bei biao ji de xi bao 。dui 12ge pei tai ban ma yu CDAjin hang huo ti shi shi cheng xiang guan cha ,ji zhong da yao zai DAde EHTguo cheng zhi hou 60 hpf(hours post fertilization)zuo you ,CDAyu wei bu jing mai fen kai ,CHTde EHTguo cheng kai shi ,yi zhi chi xu dao 156 hpf。CDAda bu fen de EHTguo cheng fa sheng zai 60 hpf-84 hpf,sui shi jian de tui yi ,EHTpin lv man man xia jiang 。bu tong shi jian duan 60-84,84-108,108-132,132-156he 156-180 hpf EHTde pin lv (bu tong shi jian duan si ge ti jie de chu ya pin lv )fen bie shi 5.60±0.68,3.40±0.81,1.50±0.40,0.80±0.58he 0.0±0.0。zai tong ji de 15xiao shi zhong ,mei si ge ti jie CDAchu ya pin lv (3.57±0.57)di yu DAchu ya pin lv (5.86±0.26)。yin ci ,CDAzai shi kong shang du li yu DA,tong guo EHTguo cheng chan sheng HSPCs。wei le jin yi bu tan suo CDAchan sheng de HSPCs,wo men li yong Tg(CD41:GFP;coro1a:Ds Red),Tg(kdrl:mem Cherry;coro1a:Kaede)zhui zong DAhe CDAgang chan sheng de HSPCsde hang wei te zheng 。gang chan sheng de xi bao zhong han you coro1a-Ds Redxin hao he CD41-GFPruo xin hao ,bing ju coro1a-Kaedexin hao ye zai gang chu ya de kdrl-mem Cherry+xi bao zhong biao da ,zhe shui ming wo men ke yi yong coro1a-Kaedeguang zhuai hua lai biao ji DAhe CDAgang chan sheng de HSPCs。jie guo xian shi ,CDAchu ya chan sheng de coro1a-Kaedexi bao da yao 30%-40%hen kuai fen lie cheng liang ge zi xi bao ,12xiao shi nei hui jin hang di er ci fen lie ,sui hou yi xie xi bao hui jin ru xie guan he xie ye xun huan ,ling yi bu fen xi bao hui bao chi yuan wei zhi jin hang di san ci fen lie 。ji zhong ,ou er neng guan cha dao yi xie xi bao jin ru xie ye xun huan hou hui zai ci jin ru CHTwei huan jing 。CDAchan sheng de xi bao zai xi bao hang wei xue shang hen xiang HSPCsde hang wei ,xing tai shang cheng qiu xing fen lie cheng qian ti xi bao ,ti ji fen lie hou hui bian xiao 。xiang bi zhi xia ,CDAchan sheng de coro1a-Kaede+xi bao yi da ban jing guo yi ci fen lie hui chan sheng sui yang de qian ti xi bao ,xi bao xing tai cheng bian xing chong xing tai er ju bian da 。jing guo 10ge xiao shi ,zhe xie sui yang qian ti xi bao hui fen lie cheng 3-4ge zi xi bao ,qian yi dao CHTwei huan jing zhong cheng xian fen zhi zhuang biao xing ,yi xie xi bao hui sui xie guan qian chu CHTbing qian yi zhi pi fu ,cheng xian dian xing de sui ji xi bao de hang wei 。da yao you 2%-5%bei biao ji de xi bao zai chu ya shi hui po sui si diao 。tong guo guang zhuai hua biao ji DAhe CDAchu ya de coro1a-Kaede+xi bao ,jing guo 60xiao shi de zhui zong ,zhe xie xi bao hang wei xue de zhui zong biao ming CDAchan sheng de HSPCscun zai zhao yi zhi xing 。you yu DAhe CDAchan sheng de HSPCszai shi kong shang he qian yi qing xiang shang you suo bu tong ,wo men tan tao ji zhong qian zai de diao kong fen zi 。Runx1dui yu DAde EHTguo cheng lai shui shi bi bu ke shao de zhuai lu yin zi ,na Runx1shi ru he ying xiang CDAou de EHTguo cheng de ne ?wo men li yong ban ma yu tu bian ti runx1w84x/w84xTg(kdrl:EGFP),fa xian CDAmo fa wan cheng EHTguo cheng ,you xie xi bao hui you chu ya de qu shi ,dan shi zui hou dou shou su hui qu 。zhe shui ming CDAhe DAyi yang ,EHTguo cheng dou xu yao zhuai lu yin ci Runx1lai diao kong 。zai zao xie guo cheng zhong ,xie liu dui xie guan jian qie ying li cu jin le zao xie gan xi bao de chan sheng 。wo men li yong mo xie liu de tnnt2aswu77ban ma yu tu bian ti ,fa xian CDAde EHTguo cheng bei yi zhi ,er ju da bu fen HSPCsxiang CDAxie guan nei ce chu ya ,er DAxiang fu ce chu ya chan sheng de HSPCs。shang shu jie guo biao ming ,cong DAdao CDAlian xu fa sheng de EHTguo cheng ,wei zheng ge dong mai dou you zao xie neng li di gong le zheng ju 。wei le jin yi bu tan jiu dong mai bu tong shi jian duan dui cheng ti ban ma yu zao xie de gong suo qing kuang ,wo men li yong Tg(kdrl:Cre ERT2;actb2:lox P-Ds Red-stop-lox P-EGFP)s928zhuai ji yin ji tong ,zai wu ge shi jian dian 20 hpf,60 hpf,72 hpf,120 hpfhe 9 dpf(days post fertilization)dui ban ma yu pei tai jin hang 4-OHT(4-Hydroxytamoxifen)chu li ,bing jian ce chu 4-OHTzhi you dao xie guan ji xie ye xi bao Crechong zu mei huo xing 。dui 4ge yue ling hou de cheng nian ban ma yu de bu tong zu zhi ou yu jin hang qie pian mian yi ying guang ran se 。li yong GFPhe Lcp1liang ge kang ti gong tong biao ji chu zao ji pei tai lai yuan de zao xie xi bao ,20 hpfchu li zu zhong ,cheng nian ban ma yu zao xie zu zhi zhong GFPyang xing xi bao zhan Lcp1+xi bao de bi li zui gao ,shen zang zhong zhan bi 76%,xiong xian zhong zhan bi 89%,sui zhao chu li shi jian de tui yi ,GFP+Lcp1+xi bao bi li xia jiang 。xiang bi yu 20 hpfchu li zu ,60 hpfchu li zu zhong GFP+Lcp1+xi bao zai xiong xian bi li xia jiang de fu du yao bi shen zang zhong de qing wei yi xie ,zhe an shi zhao zhe yi shi duan dong mai zao xie dui cheng nian xiong xian lin ba xi bao gong suo jiao da 。yu ci tong shi ,da duo shu cheng nian ban ma yu zhong nao bu xiao jiao zhi xi bao you kdrl+xi bao fen hua er lai ,zhe zhi chi le ding xiang zao xie lai yuan de xiao jiao zhi xi bao de guan dian 。zhi hou ,wo men zai Tg(kdrl:Cre ERT2;coro1a:lox P-Ds Redx-stop-lox P-GFP)s928zhuai ji yin ji tong li yong le xiang tong chu li fang fa ,de chu le shang shu xiang shi de jie lun 。yin ci ,sui zhao shi jian de tui yi ,kdrl+dong mai nei pi xi bao tong guo EHTguo cheng dui cheng nian zao xie pu ji de gong suo neng li zhu jian jian ruo 。gen ju wo men de yan jiu biao ming ,zai ban ma yu zhong wei yu CHTou de CDAzuo wei xin fa xian de zao xie wei dian ,yu AGMou de DAlei shi ,ju you tong guo EHTguo cheng chan sheng HSPCsde neng li ,bing ju chan sheng de HSPCsqian yi zhi bu tong zao xie zu zhi qi guan 。wo men de fa xian kuo zhan le ban ma yu zao xie fa yo guo cheng de li lun yan jiu 。
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