The Study On Establishment Of Gestational Diabetes Mellitus Mice Model And Experimental Intrauterine Hyperglycemia Induces DNA Methylation In Pancreas Of The Offspring Mice

Objectives:gestational diabetes mellitus(GDM),defined as a glucose intolerance resulting in hyperglycaemia of variable severity with onset or first recognition during pregnancy,is one of the most common complications during pregnancy.GDM not only has a lot of adverse effects on pregnant women,but also has some adverse effects on their offspring.A growing number of epidemiological studies have found that offspring exposed to intrauterine hyperglycaemia are at increased risk for developing future obesity,type 2 diabetes,and cardiovascular disease.However,the mechanisms responsible for these lone-term effects remain poorly understood.Therefore,this study was designed to establish reliable mice model of gestational diabetes mellitus,use the RRBS(reduced representation bisulfite sequencing)assay to detect the DNA methylation on the offspring’s pancreatic tissue,and revealing the molecular mechanisms of the increased risk of these diseases on the offspring.This study will provide a new perspective for clinical intervention for GDM patients,and to reduce and avoid the influence of intrauterine hyperglycemia on their offspring.Methods:1.Establishment of gestational diabetes mellitus mice model.Sixty female mice at 9-10 weeks randomly divided into 6 groups(5 GDM groups and 1 control group).Female mice and male mice were caged together with the proportion of 1:2.The time when the vaginal pessary was observed in female mice was recorded as pregnancy 0.5d.On the 1.5th day of pregnancy after fasting 12 h,the five GDM groups were injected with 100mg/kg(group A)or 120mg/kg(group B)or 150mg/kg(group C)or 180mg/kg(group D)or 200mg/kg(group E)of STZ once through abdominal cavity respectively and control group was injected with citrate buffer solution instead.Body weight,food and water intake,fasting blood glucose,random blood glucose,success rate,mortality rate were observed.2.Reduced representation bisulfite sequencing.Using the RRBS assay to compare the alterations in DNA methylation in pancreatic tissue of the 2nd generation mice.The average methylation level in promoter and CGI,the number and distribution of differentially methylated region and gene ontology were analyzed.Results:1.In comparison with the control mice,mice of group C,group D and group E,the body weight was significantly decreased,and the water intake,fasting blood glucose level and random blood glucose level was significantly increased.In the late trimester,mortality rates of D group and E group were 30% and 50% respectively,and the success rates of groups A and B and C and D and E were 0 and 0 and 90% and 60% and 50% respectively.2.Three kinds of cytosine methylation(CG/CHG/CHH)were mainly distributed in the promoter region and CGI.The average methylation level of the three kinds of cytosine methylation distributed in the promoter region in GDM group was higher than in normal group.The number and distribution of differentially methylated region(DMR)are different between the two groups.Gene ontology showed the DMRrelated genes were mostly relating to the process of biological regulation,developmental process,metabolic process,cell part,organelle,catalytic activity,molecular transducer activity,etc.Conclusions:1.The ideal dose of STZ for establishment of GDM mice model is about 150mg/kg by injection through abdominal cavity,which leads to higher success rate and lower death rate.2.Hyperglycemic intrauterine environment appears to be involved in DNA methylation in offspring,and may be one of the reasons of susceptibility to T2 DM,obesity,and cardiovascular disease in adult offspring.