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目的比较两种方法(血清诱导方法和无血清诱导方法)诱导小鼠胚胎干细胞分化为定形内胚层细胞的效率。方法利用血清诱导法和无血清诱导法分别诱导分化小鼠胚胎干细胞,根据定形内胚层表面标记蛋白(Cxcr4、c-Kit和E-cadherin)的表达,通过流式细胞术分析定形内胚层诱导的时间及效率,并利用RT-PCR检测两种方法诱导的定形内胚层基因表达谱;同时利用荧光定量PCR检测无血清诱导过程中内胚层基因的表达情况;利用流式细胞术分选Cxcr4和c-Kit双阳性细胞进行定形内胚层基因表达的鉴定。结果血清诱导法和无血清诱导法都能够将胚胎干细胞诱导分化为定形内胚层,其中无血清组的诱导效率高于血清组,高达74.19%,并且在诱导的第4天就能到达峰值。结论建立了高效快捷的诱导胚胎干细胞分化为定形内胚层细胞的方法,为进一步向肝、胰等组织细胞诱导打下了基础。
OBJECTIVE: To compare the efficiency of two methods (serum induction and serum-free induction) in inducing the differentiation of mouse embryonic stem cells into definitive endoderm cells. Methods Mouse embryonic stem cells were induced to differentiate by serum induction and serum-free induction respectively. The definitive endoderm-induced (Cxcr4, c-Kit and E-cadherin) expression was analyzed by flow cytometry Time and efficiency. The gene expression profiles of definitive endoderm induced by the two methods were detected by RT-PCR. Meanwhile, the expression of endoderm genes in serum-free medium was detected by fluorescence quantitative PCR. The expression of Cxcr4 and c -Kit double positive cells for the identification of definitive endoderm gene expression. Results Both serum induction and serum - free induction induced the differentiation of embryonic stem cells into definitive endoderm. The induction efficiency of serum - free group was higher than that of serum group by up to 74.19%, and reached the peak on the 4th day of induction. Conclusion A rapid and efficient method of inducing embryonic stem cells to differentiate into definitive endoderm cells was established, which lays the foundation for further inducing the cells of liver and pancreas.