【摘要】 目的利用小鼠胚胎干细胞进行体外造血细胞分化,研究LMO2蛋白在成血管细胞分化过程中的作用。方法构建小鼠成血管细胞特异性表达lmo2或绿色荧光蛋白的表达载体,分别转染小鼠胚胎干细胞。经过筛选获得细胞克隆进行自发分化以形成胚胎体。收集分化0～12 d的胚胎体细胞,检测造血细胞相关基因的表达;利用EB细胞进行细胞集落形成实验及血细胞集落形成单位实验,并统计集落形成数。结果成功构建了成血管细胞特异性表达目的基因的表达载体,转染目的基因的小鼠胚胎干细胞在其分化中产生的成血管细胞可特异表达目的基因。过量表达lmo2能够促进造血相关基因的表达,所产生细胞集落数是对照组的2倍,晚期造血分化的血细胞集落形成单位总数是对照组的2.5倍,其中红系集落单位数是对照组的3倍。结论在成血管细胞中过量表达LMO2,促进其细胞增殖及其造血细胞分化。更多还原

【Abstract】 Objective To investigate function of the Lim-only protein(LMO2)in hemangioblast generated from murine embryonic stem cells differentiation to hematopoietic cells.Methods The hemangioblast-specific expression vector with lmo2 or green fluorescence protein gene was constructed,respectively.The murine embryonic stem cells were transfected by the hemangioblast-specific expression vectors.The neomycin-resistance ES cell clones were obtained after having been screened by G418.The cell clones were spontaneously differentiated into embryo bodies(EB)containing hemangioblast.Expression of the hematopoietic genes was investigated by real-time reverse transcription-ploymerase chain reaction during EB differentiation.For the EB cells,blast-cloning forming cells analysis and blood-colony forming unit analysis were then performed,respectively.The numbers of the blasts were counted during hematopoietic differentiation.Results The hemangioblast-specific expression vector with lmo2 or green fluorescence protein was transfected into ES cells.The neomycin-resistance ES cells generated EBs from 2.5 days to 10 days.Real time reverse transcription-ploymerase chain reaction analysis indicated that overexpression of lmo2 increased the expression of hematopoietic genes(gata1,tal1,β-h1,and β-major globin)during EB formation.Blast-cloning forming cells analysis showed that the numbers of the blasts generated by ES/lmo2 was 2-or 3-fold than those in the controls.The total numbers of the blood-colony forming unit or the numbers of the erythrocyte colony-forming unit generated by ES/lmo2 were 2.5 times or 3 times,respectively,when compared with the controls.Conclusion LMO2 enhances the proliferation and differentiation of hemangioblasts.更多还原