摘要
目的 研究小鼠胚胎干细胞系ES D3在血管内皮生长因子 (VEGF)的作用下表达造血相关基因的情况。方法 先将ES D3形成拟胚体 ,将拟胚体细胞转入含不同浓度VEGF和VEGF +干细胞因子 (SCF)培养基中。分为VEGF 2 0ng/ml组、VEGF 10ng/ml组、VEGF 5ng/ml组、VEGF 5ng/ml+SCF组、VEGF 10ng/ml+SCF组、VEGF 2 0ng/ml+SCF组 ;同时设不加因子的自发分化对照组。采用逆转录 聚合酶链反应 (RT PCR)检测各组造血转录因子GATA 2和早期造血细胞阶段表达的基因c kit和 β H1表达 ,并进行定量分析。 结果 经 1周诱导培养 ,各实验组均可检测GATA 2表达 ,其中VEGF 2 0ng/ml组和VEGF +SCF组表达较强。未分化胚胎干细胞不表达c kit和 β H1mRNA ,拟胚体细胞开始表达c kit,实验组以VEGF 10ng/ml+SCF组和VEGF 2 0ng/ml +SCF组表达最强。VEGF 10ng/ml组和VEGF 5ng/ml组 ,β H1的表达分别为阴性和弱阳性 ,其他各组可检测到阳性条带 ,以VEGF 10ng/ml +SCF组表达最强 ;而自发分化组未检测到上述mRNA的表达。结论 对造血转录因子和早期造血细胞阶段表达基因的检测可进一步证实胚胎干细胞的成血分化 ,VEGF或VEGF
Objective To study the expression of genes associa te d with early hematopoietic differentiation in murine embryonic stem cells(ESC) i nduced by vascular endothelial growth factor(VEGF) .Methods ES -D3 were allowed to grow on mouse fetal fibroblast feeder layer, and then be de veloped into embryoid bodies(EB). EB cells were transferred into medium suppleme nted with different concentrations of VEGF and VEGF+SCF for one week.VEGF 20 ng/ ml group, VEGF 10 ng/ml group, VEGF 5 ng/ml group, VEGF 5 ng/ml+SCF group, VEGF 10 ng/ml+SCF group and VEGF 20 ng/ml+SCF group were designed. The group of s pontaneous differentiation without cytokine(s) was control. Hematopoietic transcription factor GATA-2 and early hematopoietic differentiation genes(c-kit and β-H1) were dete cted by RT-PCR. The relative coefficient of mRNA expression was analysed.Results ES-D3 grow and form EB at 4 day GATA-2 mRNA can be detected in every group of experiment after one week culture. It was expressed at very high levels in the group of VEGF20 ng/ml and VEGE+SCF. Undifferentiated ES cell s did not express c-kit and β-H1 mRNA. c-kit mRNA can be detected in EB c ell s.The high levels were detected in the group of VEGF 10 ng/ml+SCF and VEGF 20 n g/ml+SCF. Emergence of β-H1 mRNA expression was evident in the cells cultured with VEGF 20 ng/ml and VEGF+SCF. High expression was detected in the group of VEGF 10 ng/ml+SCF. The mRNA expression was not detected in the group of spontan eous differentiation. Conclusions Hematopoietic differentiation from ESC can be induced by VEGF. The hematopoietic character of these cells der ived from ESC is supported by the demonstration that they express genes associat ed with early hematopoietic differentiation.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2004年第3期207-209,共3页
Journal of Applied Clinical Pediatrics
