| Objective: High concentrations of glucose have adverse effects on the growth and development of mice,including diabetes,vascular and neurological malformations,and congenital diseases.Because of the complex metabolic pathways of glucose in the body,the effects on the body are also multifaceted.Previous studies mostly focused on the adverse effects of hyperglycemia on the overall development of mice,and rarely focused on target organs.This study mainly explores the effect of hyperglycemia on mouse embryonic heart development,including phenotypic changes and mechanism exploration.Through the study of embryos cultured in vitro and 13.5d embryo models,the aim is to explain how the heart of mouse embryos changes and the possible mechanism of action under high concentration of glucose.Methods: Using in vitro fertilization(IVF)and embryo culture in vitro,setting different glucose concentrations,observing the early embryonic development of mice,including the development rate of each period and the expression of key genes in the blastocyst stage.Because it is difficult to achieve gene knockdown and overexpression on embryos,this experiment chose to study the hyperglycemia(27.5m M)by knockdown and overexpression of target genes in mouse ovarian granulosa cells(GRM02)at the cellular level.)Phenotypic changes and mechanism of action on mouse embryonic heart development.Use stereo microscope to detect mouse embryonic pre-implantation stage development and related gene expression through in vitro culture,immunofluorescence,q RT-PCR,etc.;through mouse 13.5d genomic DNA/RNA extraction and bisulfite determination(BSP),immunohistochemistry,etc.to detect 13.5d mouse embryo methylation,cardiac phenotype,key gene expression;through the immunofluorescence of cell GRM02 and Plakoglobin knockdown,detect the expression of related genes after knockdown.Results: The study of mouse preimplantation embryo development showed that under high glucose conditions,mouse early embryonic development was significantly inhibited,which was mainly reflected in the significant decrease in the degree of blastocystization and changes in the expression of related genes.Under high glucose conditions,The expression of GATA4 and Tbx2 in mouse preimplantation embryos increased significantly.The transplanted embryos developed to 13.5 days and then carried out in vitro studies.The results showed that the embryos in the high glucose group were more difficult to implant,and the incidence of malformations was higher when they developed to 13.5 days;compared with the control group,under high glucose conditions The expression levels of Plagl1 and Plakoglobin in the 13.5d embryos showed an upward trend,and the heart development-related genes GATA4 and Tbx2 were also differentially expressed;compared with the control group,the Plagl1 promoter region of the high-sugar embryos was hypermethylated.Using STZ-induced high glucose mouse model,neonatal mice can be produced after normal co-cage.The neonatal hearts of diabetic mother mice show obvious abnormalities.The results of HE staining show the abnormal development of the hearts of the experimental group,which can affect Plakoglobin in GRM02 cells.After knocking down,the expression level of Plagl1 decreased simultaneously;and after overexpression of Plakoglobin in the cell,the expression level of Plagl1 increased significantly.Conclusion: This study shows that during the preimplantation and intrauterine development of the embryo,high glucose will have a significant impact on mouse embryo and heart development.High glucose can increase the expression of the maternal imprinting gene Plagl1,and its possible upstream gene Plakoglobin also shows the same trend of change.Since these two genes also play an important role in the development of the embryonic heart,this study suggests that plagkoglobin’s regulation of plagl1 expression may play a key role in the effect of high glucose on mouse embryonic heart development. |
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