ABSTRACT
BACKGROUND: Insulin like growth factor 2 (IGF2) is an important embryonic mitosis growth promoting factor, whichplays a critical role in the process of maintaining normal cell growth. The gene expression of IGF2 is under epigeneticregulation in the way of genomic imprinting. Imprinting loss of IGF2 is likely to be associated with the abnormaldevelopment of the individual and tumorigenesis.OBJECTIVE: To investigate the effect of imprinting loss of IGF2 gene on the differentiation of mouse embryonic stemcells into islet-like cells.METHODS: Two kinds of mouse embryonic stem cells (SF1-G and SF1-1) were induced to differentiate into islet-likecells in vitro. The expression of Insulin gene was detected by real-time PCR and cell immunofluorescence. Theexpression of IGF2 was detected by the polymerase chain reaction-restriction fragment length polymorphism in the cellsbefore and after induced differentiation. The level of insulin released at terminal differentiation stage was tested by insulinrelease assay in vitro.RESULTS AND CONCLUSION: (1) There was no change in the imprinting state of the two kinds of mouse embryonicstem cells with normal and imprinted IGF-2 gene before and after differentiation. (2) In the induced cells, the expressionlevel of insulin in the SF1-1 group was higher than that in the SF1-G group, although there was no significant differencein the insulin release between the two kinds of cells. (3)The imprinting loss of IGF-2 gene may be related to theup-regulation of insulin mRNA expression in terminal cells during induced differentiation.
ABSTRACT
Objective To investigate the molecular regulatory mechanism of glucagon like peptide 1 (GLP-1) on proliferation, differentiation and apoptosis of human umbilical cord blood endothelial progenitor cells (EPCs). Methods EPCs were isolated from the umbilical cord blood of healthy pregnant women and cultured in 6-hole cell plate at 2×105 density in vitro, transfected with empty vector plasmid (control group), pcDNA3-GLP-1 plasmid (GLP-1 group), pcDNA3-GLP-1plasmid+AMD3100 (GLP-1+AMD3100 group) and simple AMD3100 (AMD3100 group). The pcDNA3-GLP-1 was transfected into EPCs. The 25μmol/L AMD3100 was used to block the SDF-1/CXCR4 signal pathway of EPCs for 1 h. The cell proliferation was determined by MTT method. The mRNA expressions of differentiation and apoptosis related genes PPARγ, C/EBPα and Caspase-3 were investigated by RT-PCR, and Caspase-3 activity was determined by Caspase-3 activity assay kit. Results Compared to control group, AMD3100 inhibitor showed no effects on cell proliferation, differentiation and apoptosis, while over-expression of GLP-1 in EPCs obviously promoted cell proliferation, and differentiation related genes PPARγand C/EBPαmRNA expression, but down-regulated mRNA expression and the activity of Caspase-3 significantly (P＜0.05), indicating that GLP-1 increased proliferation and differentiation of EPCs while decreased cell apoptosis. When the SDF-1/CXCR4 signaling pathway was blocked by AMD3100, over-expression of GLP-1 induced promotion of cell proliferation, and the differentiation was decreased significantly and the apoptosis was significantly increased (P＜0.05). Conclusion These data confirm that GLP-1 might promote EPCs proliferation and differentiation, and inhibit cell apoptosis through the regulation of the SDF-1/CXCR4 signaling pathway.
ABSTRACT
Objective To observe the effects of insulin-like growth factor-2 (IGF2) in the course of mouse embryon-ic stem cells induced to differentiate into islet-like cells. Methods Mouse ES cells were induced to differentiate into islet-like cells in vitro. The expression of islet specific markers was tested by RT-PCR and immunofluorescence assay. RT-PCR/RFLP was used to test the imprinted genes IGF2 parental expression in cells at different stages. Results Islet specific mark-ers were expressed in differentiated cells, such as insulin, glucagon and C-peptide. PCR-RFLP showed that imprinted genes IGF2 derived from embryonic stem cells were biallelic expression and loss of imprinting. Conclusion Gene imprinting sta-tus of IGF2 was changed in differentiated cells in vitro.