Effect of the imprinting loss of insulin like growth factor 2 gene during the differentiation from mouse embryonic stem cells to islet-like cells in vitro

doi:10.3969/j.issn.2095-4344.2017.09.010

Abstract

Abstract:

BACKGROUND: Insulin like growth factor 2 (IGF2) is an important embryonic mitosis growth promoting factor, which plays a critical role in the process of maintaining normal cell growth. The gene expression of IGF2 is under epigenetic regulation in the way of genomic imprinting. Imprinting loss of IGF2 is likely to be associated with the abnormal development of the individual and tumorigenesis.
OBJECTIVE: To investigate the effect of imprinting loss of IGF2 gene on the differentiation of mouse embryonic stem cells into islet-like cells.
METHODS: Two kinds of mouse embryonic stem cells (SF1-G and SF1-1) were induced to differentiate into islet-like cells in vitro. The expression of Insulin gene was detected by real-time PCR and cell immunofluorescence. The expression of IGF2 was detected by the polymerase chain reaction-restriction fragment length polymorphism in the cells before and after induced differentiation. The level of insulin released at terminal differentiation stage was tested by insulin release assay in vitro.
RESULTS AND CONCLUSION: (1) There was no change in the imprinting state of the two kinds of mouse embryonic stem cells with normal and imprinted IGF-2 gene before and after differentiation. (2) In the induced cells, the expression level of insulin in the SF1-1 group was higher than that in the SF1-G group, although there was no significant difference in the insulin release between the two kinds of cells. (3)The imprinting loss of IGF-2 gene may be related to the up-regulation of insulin mRNA expression in terminal cells during induced differentiation.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Embryonic Stem Cells, Genomic Imprinting, Insulin-Like Growth Factor II, Insulin, Tissue Engineering

CLC Number:

R394.2

Liu Feng, Peng Yu-huan, Tang Jia-zhen, Jang Shan. Effect of the imprinting loss of insulin like growth factor 2 gene during the differentiation from mouse embryonic stem cells to islet-like cells in vitro[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(9): 1362-1367.

Figures/Tables 1

2.1 PCR-RFLP 检测印迹基因表达结果材料介绍中说明IGF2在SF1-G和SF1-1中呈现出不同的印记表达模式，实验收集未分化阶段的小鼠胚胎干细胞和诱导分化第7天的细胞，使用PCR-RFLP方法验证这2株小鼠胚胎干细胞中IGF2基因印记的实际情况。如图1所示，在未分化阶段，IGF2在SF1-G中呈现父源性表达，而在SF1-1中呈现出双等位表达情况，发生印记丢失，这个结果和小鼠胚胎干细胞背景材料一致。通过7 d的诱导分化，IGF2在SF1-G中也呈现父源性表达，而在SF1-1中呈现出双等位表达，这与未分化前的表达规律一样，说明经历了高效的诱导分化后，IGF2印记基因的表达模式并没有发生改变。 2.2 诱导获得胰岛素阳性细胞的基因表达和免疫染色分析收集诱导分化第7天的细胞进行RT-PCR分析和免疫荧光染色检测。如图2所示，诱导分化的终末细胞表达胰腺前体标记pdx1，表达胰腺β细胞标记Insulin，表达α细胞和δ细胞标记(Glucagon和 Somatostatin)，同时也表达胰淀粉酶标记Aamylase。相比诱导起始的第0天细胞而言，这些胰岛相关标记在第7天的表达水平明显上调，在2株不同的小鼠胚胎干细胞中都能得到相似的结果。免疫荧光染色结果显示，2株小鼠胚胎干细胞在第7天都能检测到Insulin蛋白的表达(图3)，说明诱导分化能够获得胰岛素阳性细胞。 2.3 Insulin基因表达量和葡萄糖刺激胰岛素分泌诱导的起始阶段(第0天)，IGF2基因在SF1-1组中的表达水平高于其在SF1-G组的表达水平，这种现象持续到了第7天，结果表明2株小鼠胚胎干细胞的IGF2印记表达差异确实引起了IGF2表达量上的差异(图4)。进一步检测Insulin的表达水平发现，第7天Insulin表达量较第0天显著上调，而且2株小鼠胚胎干细胞获得的终末阶段细胞中Insulin的表达水平差异有显著性意义(图4)，结果提示小鼠胚胎干细胞起始IGF2印记状态的差异可能导致了诱导终末细胞Insulin表达水平的差异。胰岛素释放实验用来检验诱导细胞针对葡萄糖刺激进行胰岛素分泌的能力，如图5所示，胰岛素释放量呈现明显的葡萄糖浓度剂量依赖性，在25 mmol/L葡萄糖浓度下，胰岛素释放水平是5.5 mmol/L葡萄糖浓度下的2.0-3.0倍，在不同葡萄糖浓度条件下胰岛素的释放水平呈现出显著差异。在相同的葡萄糖浓度条件下，2株小鼠胚胎干细胞的胰岛素释放量差异无显著性意义，结果显示通过7 d的诱导分化能够获得具备胰岛素释放能力的胰岛素阳性细胞，但是SF1-G和SF1-1的胰岛素释放水平没有明显差异。"

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