| Objective:This study aimed to verify the effect of overexpression and silencing expression of Caveolin-1 on the function of EPCs and the expression levels of PI3 K and AKT in vitro.This study was intended to elucidate whether Caveolin-1 is involved in the regulation of EPCs function and Whether the PI3K/AKT signaling pathway is involved in the regulation of EPCs by Caveolin-1.Methods:(1)Primary culture and identification of EPCs:EPCs were obtained from bone marrow of db/db male mice with type 2 diabetes aged 8 weeks.The layer of mononuclear cells was isolated with densitycentrifugation,and was then re-suspended in an offibronectin-coated vessel to cultivate generation.Immunofluorescence assays was used to verify the EPCs,respectively.(2)Transfection of EPCs and detection of transfection : EPCs were transfected with overexpressed lentiviral blank vector,overexpressed lentiviral vector,silenced lentiviral blank vector and silenced lentiviral vector,respectively.Therefore,the present study was divided into overexpression blank vector group,overexpression group,silent expression blank vector group,silent expression group and untransfected control group.To determine the amount of virus added to EPCs during transfection,the optimal MOI value for each group of lentiviral transfections was explored by the efficiency of lentiviral infection.Real-time quantitative PCR(qPCR)was used to detect the expression level of Caveolin-1 mRNA transfected with EPCs in each group of lentiviral vectors(including 3 silencing expression lentiviral vectors and one overexpressing lentiviral vector),in order to evaluate the transfection efficiency.The silencing of the lentiviral vector with the highest silencing efficiency was screened for subsequent experiments.(3)Detection of EPCs after transfection:After transfecting EPCs in type 2 diabetic mice with lentivirus,the proliferation,migration,adhesion and angiogenesis functions of EPCs in each group were detected,respectively.(4)Detection of PI3 K and AKT expression levels in EPCs after transfection:After transfecting EPCs in type 2 diabetic mice with lentivirus,mRNA and protein expression levels of PI3 K and AKT in EPCs of each group were detected by qPCR and Western blot,respectively.Results:(1)The positive rate of DiI-ac-LDL and FITC-UEA-I double staining was 73.50%±7.47.Immunofluorescence identification showed that the surface antigens of cultured cells were positive for CD31,CD34,CD133,CD144 and VEGFR2.The results of the identification showed that the cultured cells were EPCs.(2)In this study,the lentiviral vector screened for the highest silencing efficiency in this study was LV-Cav1-RNAi(37949-1).It was confirmed that the best MOI of the over-expression blank vector group,the over-expression group and the silent expression group was 50,and the best MOI of the silent expression vector group was 10.(3)There was no significant difference in the expression level of Caveolin-1 between the over-expressing blank vector group and the silent expression blank vector group,respectively.The expression level of Caveolin-1 mRNA in the overexpressing group EPCs was 4.13 times higher than that in the overexpression blank vector group(P<0.05).The mRNA expression level of Caveolin-1 in the silent expression group EPCs decreased to 5.6% of the silent expression vector group(P<0.05).Caveolin-1 overexpressed and silenced lentiviral vectors were successfully transfected into EPCs,respectively.(4)After overexpression of Caveolin-1 in EPCs of type 2 diabetic mice,the proliferative capacity decreased to 61.9% of EPCs in overexpression vector group;The migration ability decreased to 28.5% of EPCs in overexpression vector group;Adhesion ability decreased to overexpression 92.7% of EPCs in the blank vector group;The number of tubular structures formed in angiogenesis and the length of the stem decreased to 49.9% and 54.5% of EPCs in the overexpression vector group,respectively.(5)After silencing the expression of Caveolin-1 in EPCs of type 2 diabetic mice,the proliferation ability decreased to 70.4% of the EPCs in the silent expression vector group;the migration ability decreased to 34.4% of the EPCs in the silent expression vector group;the adhesion ability decreased 80.6% of the EPCs in the blank vector group were silenced;the number of tubular structures formed in angiogenesis and the length of the stem decreased to 64.4% and 54.8% of the EPCs in the silent expression vector group,respectively.(6)There was no significant difference in the expression levels of PI3 K and AKT between the over-expressing blank vector group and the silent expression blank vector group and the control group,respectively.The mRNA and protein expression levels of PI3 K and AKT in the overexpression group were significantly lower than those in the overexpression blank vector group(P<0.05).The mRNA and protein expression levels of PI3 K and AKT in the silent expression group were significantly lower than those in the silent expression vector group(P<0.05).Overexpression and silencing expression of Caveolin-1 in EPCs reduced their PI3 K and AKT expression levels.Conclusion:(1)The expression of Caveolin-1 in EPCs of type 2 diabetic mice inhibited proliferation,migration,adhesion and angiogenesis in different degrees.(2)Overexpression of Caveolin-1 in EPCs of type 2 diabetic mice inhibited proliferation,migration,adhesion and angiogenesis in different degrees.(3)Activation of PI3K/AKT signaling pathway is positively correlated with proliferation,migration,adhesion and angiogenesis of EPCs in type 2 diabetic mice,which may be involved in the regulation of angiogenesis and other functions of EPCs in caveline-1 mice.(4)Caveolin-1 is a key protein regulating the proliferation,migration,adhesion and angiogenesis of EPCs.This provides a theoretical basis for promoting the function of EPCs and preventing cardiovascular complications in patients with type 2 diabetes. |
PDF Full Text Request