| Objective:To explore the effect of high glucose on expression of Ang2 in glomerular endothelial cells cultutred in vitro and investigate the effect of Ang2/Tie2 and vascular endothelial growth factor (VEGF) on the function of glomerular endothelial cell .Methods:(1) Murine glomerular endothelial cells line were cultured and divided into three groups: Control group, treated with normal concentration of D-glucose(5.6mmol/L), and Mannitol group ,treated with mannitol (25mmol/L) plus D-glucose(5.6mmol/L) ,HG group, treated with high concentration of D-glucose(30mmol/L).After stimulated the mouse glomerular endothelial cells 0h, 12h, 24h, 48h, 72h, 96h ,detected Ang2 mRNA by RT-PCR, and associated factor Ang1, VEGF, Tie2. (2) Ang2 eukaryotic expression vector pcDNA3.1-ANG2 , and Ang2siRNA was transfeced into conditionally murine glomerular endothelial cell line by liposome. Detected Ang2 mRNA by RT-PCR after 24h. (3) Glomerular endothelial cells were transfected with two kinds of plasmids respectively,and stimulated with high glucose after 24 hour, to observe the changes of cell proliferation by MTT (4) glomerular endothelial cells stimulated with D-glucose that cultured in transwell chamber to observe the cell migration rate .Results:(1) High glucose inhibited the expression of Ang1mRNA after 12h significantly. But, after 24h,72h, 96h the expression of Ang1mRNA increased. At 0h, 48h points, there was no significant change . Ang2 and Tie2 mRNA only upregulated at 24h, 72h, 96h .while there was no significant change compared with the control group after stimulated 0h, 12h, 48h. (2) In the high glucose group,VEGFmRNA upregulated at 24h, 72h, 96h.(3) Transfected cells with the expression vector of pcDNA3.1-Ang2 and Ang2siRNA, pcDNA3.1-Ang2 transfected group, Ang2mRNA was significantly higher than the control group, Ang2siRNA significantly inhibited the expression of Ang2mRNA in the high glucose group, the difference was statistically significant (P<0.01).(4) Transfected with plasmid pcDNA3.1-Ang2, the MTT results showed that in the high glucose group and Ang2 plasmid group ,cells proliferation was significantly faster than control group. (5) Transwell chamber results showed that in high glucose group glomerular endothelial cell migration was significantly increased.Conclusion: High glucose can cause the expression of Ang/Tie2, VEGFmRNA change in glomerular endothelial cells . Ang2 can promote glomerular endothelial cell proliferation and high glucose can promote migration. |
