小鼠体节期胚胎：定向造血的发育动力学研究

发布时间：2018-04-05 07:24

本文选题：定向造血　切入点：AGM区　出处：《中国人民解放军军事医学科学院》2010年硕士论文

【摘要】： 胚胎造血发育一直是造血干细胞(hematopoietic stem cells,HSC)生物学的研究热点,分为原始和定向造血两个阶段。后者以淋巴细胞和HSC的产生为标志。近三十年来,对淋巴系统和HSC的起源认识由胚外的卵黄囊逐渐转移到胚内的主动脉-性腺-中肾区(aorta-gonad-mesonephros region, AGM region, AGM区)。Cumano等发现:胚胎循环建立前,胚内的脏壁层而非胚外的卵黄囊可通过与基质细胞和胸腺组织块共培养在体外产生B和T淋巴细胞。然而,该结论并未在其它实验室和通过其它培养体系得以验证。与淋系的体外分化系统相比,通过移植和连续移植长期重建(4-6个月)致死剂量照射的成体小鼠的造血系统是鉴定HSC的金标准。主流观点认为小鼠E10.5的AGM区是最早自主产生HSC的位点。近期,研究者发现HSC在胚外的胎盘组织出现的时间与AGM区几乎相当,且在数量上显著多于AGM区,提示HSC的发生是多位点的。既往研究发现:小鼠E9胚胎的头部含有典型的造血祖细胞,同时还表达SCL/Tal、GATA1等造血标志。但是,该部位是否具有定向造血的潜能尚属未知。本研究的第一部分,为明确能自主产生淋系的解剖部位,我们选取小鼠E8.5胚胎(1-7体节)的卵黄囊(胚外)和脏壁层(胚内)作为实验对象,此时血液循环尚未建立。在标准的集落形成实验中,两者均能产生典型的造血集落,但类型有差异。通过OP9共培养发现:脏壁层可产生B淋巴细胞,它们表达B220、CD19和IgM。采用三步法在OP9-DL1上诱导脏壁层至第16天,可产生CD44-/CD25+幼稚T细胞前体以及CD4+/CD8-单阳性T细胞。然而,上述共培养体系均不能诱导卵黄囊产生B和T淋巴细胞。我们的研究表明:与卵黄囊相比,脏壁层具有较强的体外淋系分化潜能。本研究的第二部分,我们从髓系祖细胞、淋巴细胞、HSC三个方面比较了胚胎AGM区和头部的造血特点。首先,分离E9.5-E12.5小鼠胚胎的尾部(caudal half,简称CH,该位点在E10.5发育成AGM区)/AGM区和头部,利用Ⅰ型胶原酶消化成单细胞,按照一定的胚胎当量(embryo equivalents, ee)接种于造血集落培养体系(含有SCF、IL-3、IL-6、Epo等造血因子)。第4天观察发现CH/AGM区和头部均产生了CFU-E,第7天出现CFU-GM,继而出现CFU-Mix。我们发现E9.5小鼠头部细胞产生的造血集落多为红系且致密,而CFU-GM较少。E10.5之后,CFU-GM明显增多。AGM区的粒单系集落形态均一,呈圆形或者椭圆形,而头部的粒单系集落较大,细胞较分散,形状不规则。此外,我们还研究了CH/AGM区和头部的髓系造血祖细胞(即CFU-C)的发育动力学,发现4个时间点(E9.5-E12.5)的CH/AGM区的CFU-C的数量变化不大。相反,与E10.5相比,E11.5头部的CFU-C数量显著增加达到高峰,之后又逐渐减少。利用磁珠分选,发现AGM区和头部的CFU-C集中于Tie2+细胞群。上述结果表明:小鼠胚胎AGM区和头部均存在髓系造血祖细胞,但两者在祖细胞的类型和发育动力学等方面存在差异。之后,我们考察了CH/AGM区和头部的淋系发育潜能。我们利用OP9基质细胞共培养体系考察B细胞的分化潜能。在SCF、FL、IL-7等细胞因子的作用下,第3-4天时OP9上有细胞簇出现,随后出现显著的半贴壁细胞扩增,其形态类似淋巴细胞。第7-10天收集上述细胞进行流式细胞分析发现(代表性数据):CH诱导获得的细胞表达B220(23.0%)、CD19(25.0%);头部细胞的B220、CD19的阳性率分别为:36.7%和25.8%。上述结果表明E9.5小鼠的CH和头部细胞均具有B细胞潜能。同样,分离E9.5小鼠胚胎的CH和头部,利用磁珠分选获得Tie2+细胞,接种于OP9-DL1细胞上,在SCF、FL、IL-7等细胞因子作用下,发现OP9-DL1上有大量淋巴细胞样细胞增殖。在连续的4个时间点检测发现:随着体外诱导时间的延长(7天、13天、19天、26天),幼稚的T细胞前体(CD44-/CD25+,DN3)、较成熟的双阳性细胞(CD4+/CD8+)以及CD3+/TCRβ+的比例均逐渐增加。至第26天时(代表性数据),头部与CH的变化趋势基本一致,上述3种细胞亚群的比例分别达到20.1%、7.5%、8.2%。上述结果表明:在相同的诱导条件下,小鼠E9.5的CH和头部细胞均能够分化为T淋巴细胞,说明两个位点均具有T淋巴细胞潜能。最后,我们考察了AGM区和头部的HSC潜能。分离E12.5GFP转基因小鼠胚胎的AGM区和头部,消化成单细胞后通过尾静脉进行移植。移植后5个月,采集受体外周血进行流式细胞分析发现:AGM区(n=11)和头部(n=21)的重建比例相近(45% Vs 62%),嵌合率亦相似(49.±19.0% Vs 50.±22.1%)。上述数据表明:E12.5的AGM区和头部含有的细胞均具有长期重建致死剂量照射小鼠造血系统的能力。利用磁珠分选后移植发现,AGM区和头部的Tie2+细胞均能重建致死剂量照射受体。这些结果表明,具有体内造血活性的头部细胞表达Tie2,与同期的其它造血组织类似。为检测E12.5小鼠胚胎AGM区和头部细胞移植后的多系嵌合潜能,我们对受体外周血进行了髓系(Mac-1,Gr-1)和淋系(B220,CD3)嵌合率的检测,发现:无论是AGM区还是头部细胞移植后的阳性受体,其外周血都有相当比例的供体来源的髓系和淋系嵌合。此外,我们对部分重建受体的骨髓、脾脏、胸腺进行了检测,发现AGM区和头部来源的造血细胞均能在这些成体造血组织高比例嵌合,说明E12.5小鼠胚胎的头部和AGM区的造血细胞具有体内多系分化潜能。为验证上述造血细胞的自我更新能力,我们应用已重建受体的骨髓细胞进行二次移植,2个月后检测发现所有受体全部重建,说明AGM区和头部造血细胞具有自我更新能力。基于两个位点来源的造血细胞在体内能够自我更新和多向分化,证明小鼠E12.5的AGM区和头部均含有真正的HSC。
[Abstract]:Embryonic hematopoietic growth has been a hot spot in the biology of hematopoietic stem cells ( HSC ) , which is divided into two phases : primary and direct hematopoietic . The latter is marked by the production of lymphocytes and HSCs . In the last 30 years , the origin of lymphatic system and HSC is recognized . Cumano et al . found that it was possible to produce B and T lymphocytes in vitro by co - culture with stromal cells and thymus tissue blocks . However , this conclusion was not validated in other laboratories and by other culture systems .

In the first part of this study , we selected the yolk sac ( outer layer ) and the visceral wall ( embryo ) of mouse E8.5 embryo ( 1 - 7 ) as the experimental subject . At this time , the blood circulation has not been established . In the standard colony formation experiment , it is possible to produce B lymphocytes .

In the second part of this study , we compared the hematopoietic characteristics of the embryonic agm region and the head from three aspects of myeloid progenitor cells , lymphocytes and HSC . First , the tail part ( abbreviated as CH , which was developed into agm region at E10 . 5 ) / agm region and head were isolated from the three aspects of myeloid progenitor cells , lymphocytes and HSCs . CFU - GM and CFU - GM were observed in the head cells of E9.5 - E11.5 . CFU - C in the head of E11.5 was significantly higher than that of E10 . 5 . The CFU - C of the head was larger than that of E10 . 5 .

The results showed that both CH and head cells of E9 - DL1 had the potential of B - cell . The results showed that both CH and head cells of E9 - DL1 were able to differentiate into T - lymphocytes at the same induction conditions . The results showed that both the CH and the head cells of the mice were able to differentiate into T - lymphocytes , and the results showed that both sites had the potential of T - lymphocyte .

Finally , we investigated the potential of the HSC in the agm region and the head , and separated the agm region and the head part of the e12 . 5GFP transgenic mouse embryo , digested into single cells and then transplanted through the tail vein . After transplantation for 5 months , the collected receptor ' s peripheral blood was subjected to flow cytometry analysis . The results showed that the reconstructed ratio of the agm region ( n = 11 ) and the head ( n = 21 ) was similar ( 45 % Vs 62 % ) , and the chimerism rate was similar ( 49 . 卤 19 . 0 % Vs 50 . 卤 22.1 % ) . In order to verify the self - renewal ability of marrow , spleen and thymus , we found that all the hematopoietic cells in the head and the head of E12.5 mice had the potential of self - renewal . In order to verify the self - renewal ability of these hematopoietic cells , we found that all the recipients were self - renewing . In addition , we found that all the hematopoietic cells from both the agm region and the head had self - renewal ability .

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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