CD226分子表达于造血干/祖细胞和巨核细胞谱系及神经细胞谱系参与巨核细胞倍体化调控
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摘要
[目的]本课题旨在研究CD226分子在造血干/祖细胞和相关粒系,红系和巨核细胞谱系细胞上的表达,及造血干细胞跨胚层向神经细胞分化后,CD226分子表达的变化,探索了CD226分子在巨核细胞成熟过程中的作用及与淋巴细胞功能相关抗原-1的关系。
[方法]使用血小板生成素(thrombopoietin,TPO)和粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)分别诱导成人和胎儿CD34~+细胞向巨核细胞和粒细胞分化。使用红细胞生成素(erythropoietin,EPO)诱导胎肝单个核细胞和成人CD34~+细胞向红细胞分化。使用含TPO的无血清培养基中长期培养扩增人胎肝CD34~+细胞,将扩增细胞置于Neurobasal培养基中,诱导其向神经细胞分化。使用RT-PCR,流式细胞术和免疫组化技术分析CD226分子和淋巴细胞功能相关抗原-1(lymphocyte function associated antigen 1,LFA-1)α亚单位(CDlla)在造血过程中表达的变化。使用抗体交联技术研究CD226分子和LFA-1分子在巨核细胞倍体化过程所起的作用。使用流式细胞仪和免疫组化染色技术分析CD34~+细胞向神经细胞分化过程中,CD226分子以及造血和神经细胞相关抗原表达的变化。
[Objectives] In this study, we examined the expression of CD226 on megakaryocytic, granulocytic and erythroid lineages from hematopoietic stem cells/progenitor cells in adult and fetus and its potential role in megakaryocytic maturation. We also investgated the potential of fetal liver derived-CD34~+cells induced by TPO to proliferate and differentiate further into neural stem cells and neural cells and analyzed and expression of CD226 on theses cells during transdefferetiation from hematopoietc cells to neural cells.[Methods] CD34~+ cells isolated from adult and fetus were induced to differentiate toward the megakaryocytic lineage by thrombopoietin (TPO) and the granulocytic lineage by granulocyte colony-stimulating factor(G-CSF), respectively. Mononuclear cells from fetal liver and CD34~+ cells from adult
were induced to differentiate toward erythroid-lineage by erythropoietin (EPO). FL derived-CD34~+cells isolated with magnetic beads were incubated in serum-free medium containing TPO for 60 days with weekly changes of half of the medium. The expanded cells were incubated in neurobasal medium containing N-2 supplement. The expression of CD226 and lymphocyte function associated antigen-1 (LFA-1) (CDlla, a subunit of LFA-1 molecule, were investigated during proliferation and differentiation toward granulocyte-lineage, megakaryocyte-lineage and erythroid-lineage. The effect of CD226 monoclonal antibody (MoAb) and LFA-1 MoAb on megakaryocytes was studied with antibody cross-liking technique. We also investigated the expression of CD226 molecule, hemopoiesis related antigens and neural cell related antigens on the FL derived-CD34+cells induced to differentiate toward neural cells with flow cytometry and immunocytochemistry. [Results] CD34~+ cells from adult and fetus and TPO-induced CD41~+ cells allexpressed CD226 molecule. However, CD226 was not expressed on erythroidprogenitor cells and erythroblasts and most cells of granulocytic lineage although G-CSF induced a significant increase of the expression of CD226 on CD34~+ cells in the early period of time during their differentiation toward granulocyte-lineage. CD226 MoAb acts on megakaryocytes by inducing intracellular calcium mobilization. The expression of LFA-1 decreased significantly at late stage of differentiation and maturation of fetal megakaryocytes whereas the expression of LFA-1 on adult megakaryocytes retained at a high level. Moreover, CD226 MoAb in combination with LFA-1 MoAb can shift the ploidy of generated megakaryocytes from adult-derived CD34~+ cells to higher classes significantly although CD226 and LFA-1 MoAb
[方法]使用血小板生成素(thrombopoietin,TPO)和粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)分别诱导成人和胎儿CD34~+细胞向巨核细胞和粒细胞分化。使用红细胞生成素(erythropoietin,EPO)诱导胎肝单个核细胞和成人CD34~+细胞向红细胞分化。使用含TPO的无血清培养基中长期培养扩增人胎肝CD34~+细胞,将扩增细胞置于Neurobasal培养基中,诱导其向神经细胞分化。使用RT-PCR,流式细胞术和免疫组化技术分析CD226分子和淋巴细胞功能相关抗原-1(lymphocyte function associated antigen 1,LFA-1)α亚单位(CDlla)在造血过程中表达的变化。使用抗体交联技术研究CD226分子和LFA-1分子在巨核细胞倍体化过程所起的作用。使用流式细胞仪和免疫组化染色技术分析CD34~+细胞向神经细胞分化过程中,CD226分子以及造血和神经细胞相关抗原表达的变化。
[Objectives] In this study, we examined the expression of CD226 on megakaryocytic, granulocytic and erythroid lineages from hematopoietic stem cells/progenitor cells in adult and fetus and its potential role in megakaryocytic maturation. We also investgated the potential of fetal liver derived-CD34~+cells induced by TPO to proliferate and differentiate further into neural stem cells and neural cells and analyzed and expression of CD226 on theses cells during transdefferetiation from hematopoietc cells to neural cells.[Methods] CD34~+ cells isolated from adult and fetus were induced to differentiate toward the megakaryocytic lineage by thrombopoietin (TPO) and the granulocytic lineage by granulocyte colony-stimulating factor(G-CSF), respectively. Mononuclear cells from fetal liver and CD34~+ cells from adult
were induced to differentiate toward erythroid-lineage by erythropoietin (EPO). FL derived-CD34~+cells isolated with magnetic beads were incubated in serum-free medium containing TPO for 60 days with weekly changes of half of the medium. The expanded cells were incubated in neurobasal medium containing N-2 supplement. The expression of CD226 and lymphocyte function associated antigen-1 (LFA-1) (CDlla, a subunit of LFA-1 molecule, were investigated during proliferation and differentiation toward granulocyte-lineage, megakaryocyte-lineage and erythroid-lineage. The effect of CD226 monoclonal antibody (MoAb) and LFA-1 MoAb on megakaryocytes was studied with antibody cross-liking technique. We also investigated the expression of CD226 molecule, hemopoiesis related antigens and neural cell related antigens on the FL derived-CD34+cells induced to differentiate toward neural cells with flow cytometry and immunocytochemistry. [Results] CD34~+ cells from adult and fetus and TPO-induced CD41~+ cells allexpressed CD226 molecule. However, CD226 was not expressed on erythroidprogenitor cells and erythroblasts and most cells of granulocytic lineage although G-CSF induced a significant increase of the expression of CD226 on CD34~+ cells in the early period of time during their differentiation toward granulocyte-lineage. CD226 MoAb acts on megakaryocytes by inducing intracellular calcium mobilization. The expression of LFA-1 decreased significantly at late stage of differentiation and maturation of fetal megakaryocytes whereas the expression of LFA-1 on adult megakaryocytes retained at a high level. Moreover, CD226 MoAb in combination with LFA-1 MoAb can shift the ploidy of generated megakaryocytes from adult-derived CD34~+ cells to higher classes significantly although CD226 and LFA-1 MoAb
引文
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