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背景:血管内皮生长因子家族及其受体在血管生成和新血管形成过程中具有重要作用。近来,有关血管内皮生长因子家族对血液发生的作用受到广泛关注。目的:观察血管内皮生长因子A、血管内皮生长因子C及其受体和an-giopoietin-1(ang-1)、angiopoietin-2(ang-2)及其受体在发育第3～12周人胚胎卵黄囊血岛、主动脉-生殖腺-中肾区的表达,以了解血管内皮生长因子和angiopoietin家族在血液发生过程中的作用。设计:单一样本观察。单位:潍坊医学院,形态实验中心和解剖学教研室。对象:实验于2002-02/2004-08在潍坊医学院进行,收集流产的47例孕妇的受精龄第3～12周人胚胎标本,孕妇知情同意。方法:标本连续切片,每隔10张抽出2张切片,分别做苏木精-伊红染色和免疫组织化学染色。多克隆抗血管内皮生长因子C,fms样酪氨酸激酶,flt-4,ang-1,ang-2,Tie-2抗体以及单克隆抗血管内皮生长因子A,胎肝激酶1抗体4℃孵育过夜,羊抗鼠IgG及SABC液室温下分别孵育2h,DAB显色。分别以正常兔或鼠血清代替一抗作阴性对照。光学显微镜下观察、拍照。主要观察指标:①血管内皮生长因子A和血管内皮生长因子C及其受体在发育第3～12周人胚胎卵黄囊血岛、主动脉-生殖腺-中肾区的时空表达。②ang-1和ang-2及其受体在发育第3～12周人胚胎卵黄囊血岛、主动脉-生殖腺-中肾区的时空表达。结果:①第21～25天人胚卵黄囊血岛血管内皮细胞和造血细胞强表达血管内皮生长因子A及其受体fms样酪氨酸激酶和胎肝激酶1、血管内皮生长因子C及其受体胎肝激酶4、ang-2及其受体Tie-2蛋白,弱表达ang-1蛋白。②背主动脉和中肾于发育第4周开始表达上述各种因子及其受体,免疫阳性反应物集中在大而圆、有核的造血细胞。阳性细胞的数量到第7周到达高峰。8周以后,阳性细胞数量显著减少,到12周,几乎所有的血细胞为免疫阴性反应。③生殖腺主要在第6～8周表达血管内皮生长因子A,fms样酪氨酸激酶,flt-4,ang-1,ang-2和Tie-2蛋白,但不表达血管内皮生长因子C和胎肝激酶1。④主动脉-生殖腺-中肾区的血管内皮细胞未检测到上述各因子的表达。结论:人胚卵黄囊血岛造血细胞和内皮细胞以及背主动脉和中肾共表达多种与血管发生和血液发生相关的因子。人胚内造血发生于第4周。
BACKGROUND: Vascular endothelial growth factor families and their receptors play an important role in angiogenesis and neovascularization. Recently, the role of the vascular endothelial growth factor family in blood generation has drawn much attention. OBJECTIVE: To observe the expression of vascular endothelial growth factor A, vascular endothelial growth factor C and their receptors and angiopoietin-1 (ang-2) and angiopoietin-2 (ang-2) Embryonic yolk sac blood island, aorta - gonadal - mesangial area in order to understand the role of vascular endothelial growth factor and angiopoietin family in the process of hematopoiesis. Design: Single sample observation. Unit: Weifang Medical College, Morphology Experimental Center and Department of Anatomy. PARTICIPANTS: The experiment was carried out in Weifang Medical College from February 2002 to August 2004. 47 pregnant women of miscarriage were collected from the 3rd to 12th week of human embryo samples and the pregnant women were given informed consent. Methods: The specimens were cut into sections and 2 slices were taken out every 10 slices. The sections were stained with hematoxylin-eosin and immunohistochemistry respectively. The polyclonal anti-VEGF-C, fms-like tyrosine kinases, flt-4, ang-1, ang-2 and Tie-2 antibodies as well as monoclonal anti-vascular endothelial growth factor A and fetal liver kinase 1 Overnight, goat anti-mouse IgG and SABC solution were incubated for 2h at room temperature, respectively. Respectively normal rabbit or mouse serum instead of primary antibody as a negative control. Under light microscope observation, take pictures. MAIN OUTCOME MEASURES: ① The temporal and spatial expression of Vascular Endothelial Growth Factor A and Vascular Endothelial Growth Factor C and their receptors in the yolk sac, aorta - gonad - mesonephrotic region from the 3rd to 12th weeks of development. ② The temporal and spatial expression of ang-1, ang-2 and ang-2 receptors in the yolk sac, aorta-gonad-mesonephric region in the third to twelfth week of development. RESULTS: ① The vascular endothelial cells and hematopoietic cells of human embryonic yolk sac of the 21st to 25th day strongly expressed VEGF-A and its receptor fms-like tyrosine kinase, fetal liver kinase 1, vascular endothelial growth factor C and their Receptor fetal liver kinase 4, ang-2 and its receptor Tie-2 protein, weakly expressed ang-1 protein. ② The dorsal aorta and middle kidney begin to express the above-mentioned various factors and their receptors on the 4th week of development, and the immunoreactive reactants are concentrated on the round, nucleated hematopoietic cells. The number of positive cells peaked at the 7th week. After 8 weeks, the number of positive cells was significantly reduced, by 12 weeks, almost all of the blood cells were immunoreactive. (3) The gonads expressed vascular endothelial growth factor A, fms-like tyrosine kinases, flt-4, ang-1, ang-2 and Tie-2 proteins mainly from the 6th to the 8th week but did not express vascular endothelial growth factor C Hepatic kinase 1. ④ aorta - gonad - mesothelial cells in vascular endothelial cells were not detected in the expression of the above factors. CONCLUSION: Human embryo yolk sac blood island hematopoietic cells and endothelial cells as well as dorsal aorta and mesonephros coexpress a variety of factors associated with angiogenesis and hematopoiesis. Human embryo hematopoiesis occurs in the 4th week.