小鼠胚胎E10～E12造血扩增部位的探寻及其分子作用机制的研究

发布时间：2018-04-25 12:34

本文选题：小鼠胚胎 + AGM时期　；参考：《华中科技大学》2009年博士论文

【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。
[Abstract]:Part one
[Objective] to explore the anatomical structure of hematopoietic sites in AGM (E10 ~ E12) mice.
[Methods] the female and male mice were fed with 3: 1 cage to observe the mouse embryos of the exact pregnancy by observing the mouse vaginal suppository, collecting the E10, E10.5, E11, E11.5 and E12 mouse embryos, removing the placenta and placenta, using 4% formaldehyde to fix the 3h, the ethanol was dehydrated, the paraffin was embedded and stained through the median sagittal plane of embryo fetal, and HE staining was carried out under the microscope. Under the microscope, the microscope was continuous under the microscope. Under the microscope, the microscope was continuous under the microscope. Under the microscope, continuous microscope was continuous under microscope. Under microscope, continuous microscope was continuous under microscope. Under microscope, continuous microscope was continuous under microscope. Under microscope, continuous microscope was continuous under microscope. Under microscope, continuous microscope was continuous under microscope. Under microscope, continuous microscope was continuous under microscope Observe the structure of the hematopoietic tissue.
[results] the median sagittal section of the embryo, from E10 to E12, did not see the typical gonad on the dorsal side of the abdomen, that is, the cavity tubular structure of the middle kidney and the aorta, but found that the heart developed at the time of E10, showing a circular sphere, and the internal structure of the endothelium of the myocardium and the surface of the endothelial cells, which could be seen in the E10 mouse embryo. A small number of cells in the vascular lumen and heart can be seen in the fetus. There is no obvious change in the morphology of the cardiac tissue section of.E11 with abundant cytoplasmic hematopoietic stem cells than that of E10.5. However, the number of hematopoietic cells in the heart cavity increases significantly at this time, and it is easy to see the contact between the hematopoietic cells and the inner heart endothelial cells. With the development of the embryo, the heart is developed. The shape of the internal reticular formation is gradually reduced, and the internal reticular structure is gradually reduced, and the E12 has the characteristics of the adult heart and becomes a cavity structure. The fetal liver of the mouse E10 embryo is developed at first, which is a solid structure, which is made up of the fetal liver cells closely connected, and the internal cavities are few. With the development of the embryo, the blood vessels in the fetal liver increase.
[Conclusion] the typical AGM region structure was not found through continuous slicing in the median sagittal plane of E10 to E12 embryos and HE staining. It may be related to the limitation of the tissue section. At first, the heart had a complex spatial network structure and a layer of endothelial cells covered the surface of the cardiac myocytes, which formed a huge surface during the development of hematopoietic stem cells. Product.
Part two
[Objective] to study the characteristics of mouse embryonic heart endothelial cells during E10 ~ E12 and the molecular mechanism related to hematopoietic development.
[Methods] the tissue section of E10 ~ E12 mouse embryos was immunohistochemical, and the blood vessel specific marker CD34 protein was detected by the embryo heart. By using the prepared paraffin section, the expression level of Notch signaling pathway related molecules in the fetal heart and fetal liver, the expression level of Notch 1 and Jagged1 protein were detected by immunohistochemistry. C-kit positive cells were selected by immunomagnetic beads. The expression of Notch 1 protein on the surface of the cells was detected by immunofluorescence. The embryonic heart of E10, E10.5, E11, E11.5 and E12, fetal liver and AGM region tissues were prepared by mechanical milling, collagenase digestion and filtration to prepare single cell suspension. The differential adherence method and adherent culture were used. The stromal cells were observed, the morphology of adherent cells in each tissue was observed, and the expression of CD31 was detected.
[results] the cardiac myocytes and the endothelial cells on the surface of the mouse embryonic heart of E10 ~ E12 all express the vascular specific marker CD34 protein, and the expression of E11 is the highest, while the circulating hematopoietic cells can also express most of the CD34 antigens. The immunohistochemical study of Notch signal in the tissue sections has been carried out to detect the cardiac muscle fine of E10. The hollow reticular formation of endothelial cells on the cell and its surface all express Jagged1 protein and Notch 1 protein. The expression level of the former is higher than that of the latter, with the highest level at E10.5, and with the development and morphologic changes of the heart, the expression level gradually decreases, and it is almost disappeared at E12. It is found that the vascular endothelial cells also express Jagged1, but not at the same time. Expression of Notch1 protein. C-kit positive cells were separated by the immunomagnetic beads of c-kit monoclonal antibody. By immunofluorescence, the expression of Notch 1 protein on the surface of the group was revealed. The primary cardiac parietal cells were composed of large volume, rich cytoplasm, irregular cardiomyocytes, elongated spindle shaped, less cytoplasmic endothelial cells, and easy to see the heart. The endothelial cells were attached to the surface of the cardiac myocytes, showing a uniform elongated spindle arrangement and expression of CD31 antigen. The morphology of the fetal liver adherent cells was relatively uniform, both of which were larger and rich in cytoplasm. At the same time, two cell groups were also found in the primary cells, including the larger cell adherent cells and spindly spindle endothelial cells in the AGM area. The morphology is similar to that of the cardiac parietal cells.
[Conclusion] the cardiac myocytes and endothelial cells of the heart from E10 to E12 all express CD34 antigen, and possess the characteristics of vascular endothelial cells, the reticular structure constitutes a huge surface area, and the high expression of Jagged1 and Notch 1 proteins to form a good microenvironment for embryo hematopoiesis, which may be involved in the development and expansion of hematopoietic stem cells at this time.
The third part
[Objective] to investigate the level of Notch signaling pathway in hematopoietic related parts from]E10 to E12 and to explore the location of HSCs amplification in the middle stage of embryo.
[Methods] the expression and change levels of E10, E10.5, E11, E11.5 and E12 cardiac endothelial cells and fetal liver stromal cells Notch 1 and Jagged1 were continuously observed by real-time quantitative PCR. The ratio of c-kit+ cells in the heart and fetal liver at each time point was detected by flow single standard. The E11 cardiac suspension cells after the culture of 24h were placed in the same period of cardiac endothelial cells, respectively. In the co culture system of fetal liver stromal cells and AGM region stromal cells, the ratio of c-kit+ cells was detected by 0h, 24h and 48h, and the effect of matrix cells on the amplification of HSCs was studied.
[results] the expression level of Jagged1 and Notch 1 genes in the passages and primary cells did not change in spite of the influence on the morphology of the cardiac parietal cells. We found that the Jagged1 gene of the heart adherent cells was the highest in E10.5, compared with the E10 gene level, which was (5.46 + 2.2) times of that of E10, and the expression of E11 decreased, almost the same as E10. But the expression of Jagged1 in E11.5 and E12 was very low, which was E10 (0.15 + 0.44) times and (0.12 + 0.50) times respectively. But the expression of Jagged1 gene in fetal liver stromal cells, except E11, was similar to that of E10's heart endothelial cells. All the other embryonic stages were low level expression. Through flow cytometry, we found that the embryonic heart was the heart of c-kit+ cells. The ratio of c-kit+ cells to fetal liver was almost identical, and the number of c-kit+ cells increased significantly at the time of E11, up to (12.6 + 3.2)% and (9.6 + 2.8)% respectively, but the c-kit+ cells in the heart decreased significantly at the time of E12, only (3.4 + 1.2)%. At this time, c-kit+ cells in fetal liver (11.6 + 4.1)%.E11 were co cultured 24h with cardiac endothelial cells and AGM region matrix, and the number of c-kit+ cells The amount of c-kit+ cells in the fetal liver stromal cells decreased significantly, and in co culture of 48h, the c-kit+ cells in the three groups were all low, and there was no significant difference between the groups.
[Conclusion] the expression of Jagged1 gene in the cardiac endothelial cells is changed and the expression is highest at E10.5. The development of HSCs in fetal liver is not the main function of the amplification of.Notch signal pathway with the number of c-kit+ cells. C-kit+ cells are co cultured with a variety of simultaneous stromal cells, and the E11 embryo heart and AGM region have HSCs amplification. Environment, fetal liver of E11 may not be an important part of hematopoietic expansion.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

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