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论著:二维法和三维法诱导小鼠胚胎干细胞定向分化为内皮细胞
Differentiation of Murine Embryonic Stem Cells into Endothelial Cells Using Two Dimensional Method and Three Dimensional Method
高斌 符伟国 竺挺 张祥满 董智慧 王玉琦
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作者单位:复旦大学附属上海市第五人民医院普外科
中文关键字:胚胎干细胞;内皮细胞;细胞分化
英文关键字:Embryonic stem cell; Endothelial cell; Cell differentiation
中文摘要:目的:比较二维法和三维法体外诱导小鼠胚胎干细胞(embryonic stem cell,ESC)分化为高纯度内皮细胞(endothelial cell, EC)的效率。方法:分别用二维法和三维法体外诱导小鼠ESC向EC定向分化,以胎肝激酶 1(fetal liver kinase 1, Flk 1)作为标志物进行流式分选;增加血管内皮生长因子(vascular endothelial growth factor, VEGF)浓度并对
阳性细胞加以人工分选以进一步纯化。评估两种方法诱导的分化效率,并采用反转录 聚合酶链反应(reverse transcription polymerase chain reaction,RT PCR)、免疫荧光法及DiI标记的乙酰化的低密度脂蛋白(DiI Ac LDL)吞噬试验等方法来评估ESC来源的EC(ESDEC)的生物学行为。结果:二维法流式分选的Flk 1阳性细胞比例显著高于三维法(P<0.05)。Flk 1阳性细胞在二维培养体系中呈现鹅卵石样的内皮样细胞和条索状的平滑肌样细胞,经人工分选和纯化后可得到高纯度的形态单一的并可传代培养的ESDEC。两种方法分化的ESDEC均可形成管状结构、表达EC特异性细胞表面标志物,并且DiI ac LDL吞噬实验阳性。结论:两种方法均可诱导小鼠ESC定向分化为EC,二维法更为简单有效。增加VEGF浓度并对Flk 1阳性细胞进行人工分选有助于纯化ESDEC。
英文摘要:Objective: To compare the efficiency of two dimensional (2D) method and three dimensional (3D) method in the induction of differentiation of murine embryonic stem cells (ESCs) toward endothelial cells (ECs). Methods:Both 2D method and 3D method were used to induce the differentiation of murine ESCs toward ECs. Supplementation of vascular endothelial growth factor (VEGF) and isolation of fetal liver kinase 1 positive (Flk 1+) cells were done for further purification. Biological characteristics of embryonic stem cell derived endothelial cells(ESDECs) were confirmed by reverse transcription polymerase chain reaction (RT PCR), immunofluorescence test, DiI labeled acetylated low density lipoprotein (DiI Ac LDL) uptake assay. Results: The proportion of Flk 1+ cells acquired using 2D method was significanly higher than using 3D method (P<0.05). In the 2D culture system, Flk 1+ cells exhibited the characteristics of endothelial like cells with cobblestone morphology and striated smooth muscle like cells. After further purification by manual selection, those cells displayed the morphology of endothelial cells with high purity and they were able to be subcultured. The ESDECs acquired through both methods expressed endothelial cell specific markers, formed tube like channels and incorporated DiI Ac LDL. Conclusions: Both 2D method and 3D method can be used to induce the differentiation of murine ESCs into ECs in vitro, but the former is more efficient than the latter. Supplementation of vascular VEGF and isolation of Flk 1+ cells can help to improve the purification of ESDECs.
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