【作者】 黄丽芳；

【导师】 刘文励；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 第一部分[目的]探讨AGM时期(E10～E12)小鼠造血部位的解剖结构。[方法]将雌、雄小鼠以3: 1合笼喂养,观察小鼠阴道栓获得准确孕期的小鼠胚胎,收集E10, E10.5, E11, E11.5和E12小鼠胚胎,去掉胎膜和胎盘,用4%甲醛固定3h后,进行乙醇脱水,石蜡包埋,通过胚胎正中矢状面组织切片,进行HE染色,在显微镜下连续观察造血部位的组织结构。[结果]胚胎正中矢状面切片,自E10到E12,在腹部背侧并没有见到典型的性腺,即中肾和主动脉构成的空腔管状结构,却发现在E10时心脏已经发育,表现为圆球形,内部由心肌细胞及表面的内皮细胞构成网状的空间结构,可以看到E10小鼠胚胎已经建立循环,血管管腔和心脏内部可见少量胞体较大、胞浆丰富的造血干细胞。E11的心脏组织切片形态较E10.5并无明显变化,但此时期心腔内的造血细胞数量明显增多,容易见到造血细胞与心脏内皮细胞接触。随着胚胎的发育,心脏的形态很快变化,内部网状结构逐渐减少,在E12已具备成体心脏的特点,成为空腔性结构。小鼠E10胚胎中胎肝已经发育,起初为实质的结构,由胎肝细胞紧密联系而构成,内部管腔少,随着胚胎的发育,胎肝内部血管增多。[结论]通过E10～E12胚胎正中矢状面连续切片和HE染色,并未发现典型的AGM区结构,可能与组织切片的局限性有关;心脏起初内部呈复杂的空间网状结构,且心肌细胞表面覆盖有一层内皮细胞,在造血干细胞发育过程中构成了巨大的表面积。第二部分[目的]研究E10～E12期间小鼠胚胎心脏内皮细胞特性及与造血的发育相关的分子机制。[方法]将E10～E12小鼠胚胎的组织切片做免疫组化,检测胚胎心脏是否表达血管特异性标志CD34蛋白;利用已制备好的石蜡切片,通过免疫组化检测各时间点胚胎心脏和胎肝中Notch信号通路相关分子,Notch 1和Jagged1蛋白的表达水平;并且用免疫磁珠分选出c-kit阳性细胞,通过免疫荧光的方法检测其细胞表面Notch 1蛋白的表达情况。分别取出E10, E10.5, E11, E11.5和E12的胚胎心脏、胎肝和AGM区组织,经机械碾磨、胶原酶消化和过滤制备单个细胞悬液,采用差异贴壁法及贴壁培养获得基质细胞,观察各组织贴壁细胞形态,检测CD31表达情况。[结果] E10～E12的小鼠胚胎心脏的心肌细胞及其表面的内皮细胞均表达血管特异性标志CD34蛋白,以E11表达强度最高,同时可以见到循环的造血细胞也大多表达CD34抗原。对组织切片进行了Notch信号通路免疫组化的研究,发现E10心脏心肌细胞及其表面的内皮细胞形成的中空的网状结构都表达Jagged1蛋白和Notch 1蛋白,前者表达水平高于后者,以E10.5时水平最高,且随着心脏的发育和形态改变,表达水平逐渐下降,到E12时几乎消失。同时发现血管内皮细胞也表达Jagged1,但是不表达Notch1蛋白。用c-kit单抗的免疫磁珠分选出c-kit阳性细胞,通过免疫荧光,显示这群细胞表面均匀的表达Notch 1蛋白。原代心脏贴壁细胞由体积较大、胞浆丰富、呈不规则形的心肌细胞和细长梭形、胞浆较少的内皮细胞构成,容易看到心脏内皮细胞贴附在心肌细胞表面,呈均一的细长梭形排列,表达CD31抗原。胎肝贴壁细胞形态相对均一,均为胞体较大、胞浆丰富的细胞。同期AGM区贴壁细胞在原代细胞中也可见两个细胞群体,包括胞体较大的贴壁细胞和细长梭形的内皮细胞,形态与心脏贴壁细胞相似。[结论]胚胎E10～E12期间心脏的心肌细胞及其内皮细胞均表达CD34抗原,具备血管内皮细胞特性,网状结构构成巨大的表面积,并且高表达Jagged1和Notch 1蛋白,形成胚胎造血良好的微环境,可能参与此时期造血干细胞的发育和扩增。第三部分[目的]E10～E12时期造血的相关部位Notch信号通路的变化水平及对胚胎中期HSCs扩增部位的初探。[方法]通过实时定量PCR连续观察E10, E10.5, E11, E11.5和E12心脏内皮细胞和胎肝基质细胞Notch 1及Jagged1表达及变化水平。用流式单标检测各时间点心脏和胎肝中c-kit+细胞比率。将培养24h后的E11心脏悬浮细胞分别置入由同期的心脏内皮细胞、胎肝基质细胞和AGM区基质细胞构成的共培养体系中,于0h, 24h和48h分别检测c-kit+细胞比率,研究各组织基质细胞对HSCs扩增的影响。[结果]尽管传代对心脏贴壁细胞形态有影响,但是传代和原代细胞中Jagged1和Notch 1基因的表达水平没有变化。我们发现:心脏贴壁细胞的Jagged1基因在E10.5到最高,跟E10的基因水平相比较,为E10的(5.46±2.2)倍,E11时的表达下降,几乎与E10相同,但是E11.5和E12时心脏内皮细胞Jagged1表达均很低,分别为E10的(0.15±0.44)倍和(0.12±0.50)倍。但是胎肝基质细胞Jagged1基因除E11的表达与E10的心脏内皮细胞相近外,其余胚胎时期均呈低水平表达。通过流式检测c-kit+细胞我们发现,胚胎期心脏和胎肝c-kit+细胞比率几乎一致,E11时c-kit+细胞明显增多,分别高达(12.6±3.2)%和(9.6±2.8)%,但E12时心脏中c-kit+细胞明显减少,仅为(3.4±1.2)%,此时胎肝中c-kit+细胞为(11.6±4.1)%。E11的悬浮细胞与心脏内皮细胞和AGM区基质共培养24h时,c-kit+细胞数量保持稳定,但在胎肝基质细胞中c-kit+细胞数量明显减少,在共培养48h,三组中的c-kit+细胞均呈较低水平,组间没有显著性差异。[结论]心脏内皮细胞中Jagged1基因表达是变化的,在E10.5时表达最高,伴随着c-kit+细胞数量的扩增。Notch信号通路在胎肝中HSCs的发育并不起主要作用。通过c-kit+细胞分别与各种同时期基质细胞共培养,推测E11胚胎心脏和AGM区具备HSCs扩增的环境,E11的胎肝可能并不是造血扩增的重要部位。更多还原

【Abstract】 PartⅠ[Objective] To investigate the anatomic structure of mouse hematopoietic region during AGM period of E10-E12.[Methods] Matings for embryo generation were timed and the day of vaginal plug detection was designated as embryonic day 0 (E0). The mouse embryo of E10, E10.5, E11, E11.5 and E12 were collected. The embryoemma and the placenta were removed. After fixed in formalin for 3 hours, dehydrated by gradient alcohol, embedded in paraffin according to standard protocols, sliced through sagittal plane and dyed by HE, the histological anatomy of the mouse hematopoietic region was observed under a microscope.[Results] The typical tubiform structure constituted by the aorta, gonad and mesonephron region was not observed in the dorso- abdominalis through the sagittal plane from E10 to E12. The embryo heart had developed, presenting the globular appearance with cancellous spatial structure consisted of cadiocyte and superficial slender endotheliocyte linked with each other at E10. The circulation had established at E10 from the slice of the mouse embyo. It is evident that a few cyloid mononuclear HSCs with the affluent kytoplasm had emerged in the heart and the blood vessel. There was no significance difference in the histological section of embryo heart between E10.5 and E11. But the quantity of hematopoietic cells was obviously increased at El 1, and they were easily seen to touch with the endothelitcytes of blood vessel and the heart. As the embryo developed, the shape of the heart changed quickly, with network structure diminished gradually. And at E12 the heart had becomed to the hollow organ, owning the characteristic of adult heart. The fetal liver had developed at E10, which was the substantial structure and composed with fetal liver cells arranged tightly. The blood vesse of fetal liver interior increased gradually, as the fetal liver developed. [Conclusion] The typical AGM region was not discovered through serial sections of mouse embryo’s median sagittal plane and HE staining. The embryo heart emerged as complicate network structure at E10, with the endotheliocytes covering the cadiocytes, which may provide the favorable miroenvironment for the development and proliferation of HSCs through the gigantic surface area.PartⅡ[Objective] To research the characteristics of endotheliocyte in the mouse embryo heart during E10～E12 and molecule mechanism related to the hematopoietic development. [Methods] The histological sections of E10 to E12 were immunostained with anti-mouse CD34, Notch1 and Jagged1, and examined under a microscope. The c-kit+ cells in the heart and fetal liver were sorted through immunomagnetic beads and the expression of Notch1 protein was detected by immunofluorescence technique. The mononuclear cells (MNC) of the heart, fetal liver and AGM region from E10 to E12 were respectively separated by grinding gently, digestion by collagenase and filtering through the grit. The stromal cells of above region were obtained by discrepancy adherence, and was observed under a micorcsope.[Results] The cadiocyte and the superfic endotheliocyte of E10 to E12 both expressed the CD34 antigen (the surface marker of blood vessel), with the highest expression at E11. Meanwhile the hematopoietic cells in the circulation also expressed the CD34 protein. Jagged land Notch1 protein was expressed on all stromal cells in the spherical netword strutere of the heart, the former higher than the latter. Their expressions were strongest at E10.5, and gradually decreased as the embryo developed until E12. The vascular endothelial cells expressed the Jagged1 protein, but not Notch1. Notch1 protein was dispersed homogeneously in the surface of c-kit+ cells separated by immunomagnetic beads. The adherent cells from the embryo heart of E10～E12 were constituted by the difform cadiocyte with affluent kytoplasm and the slender endotheliocyte, which arranged in paralleled regularly and was seen easily to stick on the cadiocytes. CD31 antigen was expressed in the adherent of the heart, but not in the stromal cell of fetal liver from E10～E12, which showed uniform morphous with affluent endochylema. The stromal cells from AGM region were similar to the adherent cells of the heart.[Conclusion] The cadiocytes and the endotheliocyte from the embryo heart of E10 to E12 both expressed the CD34 antigen, indicating they had the characteristics of vascular endothelial cells. And the network structure constituted by the cadiocytes and the endotheliocyte with high expression of Jagged1 and Notch1 provided the favourable microenvironment for embryo hematopoiesis, inferring that the heart could participate in the development and amplification of HSCs.PartⅢ[Objective] To investigate the expression level of Notch signal pathway in the hematopoietic related region and explore preliminarily the position for HSCs’ expansion during E10 to E12.[Methods] The gene expression of Notch1 and Jagged1 were respectively detected by real-time PCR in the stromal cells from the heart and fetal liver during E10 to E12 continuously. The rate of c-kit+ cells was monitored the BD-LSR flow cytometer from E10 to E12. The suspension cells of the heart at E11 were respectively cultured in co-culture system based in the stromal cells from the heart, the fetal liver and AGM region with the same conceptus age, and c-kit+ cells were analyzed by flow cytometer, estimating the effect of different stromal cells on HSCs’ amplification.[Results] The gene expression level of Jagged1 and Notch1 was unchanged between primary cells and passage cells of adherent cells from the embryo heart, despite the passage made some difference to the morphous of adherent cells. Compared to the E10, the Jagged1’s expression of E10.5 reached the maximum with 5.46±2.2 fold, but decreased at E11 with the same to E10. The Jagged1 ’s level of adherent cells from the heart at E11.5 and E12 were very low with 0.15±0.44 and 0.12±0.50 respectively to E10. The Jagged1 gene level of stromal cells from fetal liver during E10～E12 were low level except for E10. The rate of c-kit+ cells in the heart increased gradually from E10 with the highest level of 12.6±3.2% at E11, which was nearly similar to the fetal liver, except for E12 when the c-kit+ cells was 3.4±1.2% in the heart compared to the fetal liver with 11.6±4.1% c-kit+ cells. The rate of c-kit cells in the suspension cells from the heart at E11 maintained stable after 24h culture based in the stromal cells of the heart and AGM region with the same conceptus age, but the rate of c-kit cells obviously decreased in the condition of the stromal cells from the fetal liver. The c-kit cells of the three conditions were all at low level at 48h without significant difference.[Conclusion] The expression level of Jagged1 gene in the adherent cells from the heart was changed during E10 to E12, and reached the highest level at E10.5, with the expansion of HSCs concomitantly. Notch signal pathway may not play the important role in HSCs development in the fetal liver. It was inferred that the embryo heart and AGM region of E11 may contribute to HSCs amplification.更多还原