The Influences And Related Mechanisms Of Different Levels Of Glucose And Insulin On The Formation Of Trophoblast Cell’s Microvilli

Objective:Gestational diabetes mellitus is,now,a common obstetrical disease,of which incidence of morbidity increases year by year and can easily lead to adverse pregnancy outcome and near and future complications of mothers and offsprings.Placenta,an important organ,can maintain maternal pregnancy and fetal development during pregnancy.The structure and functional integrity of the syncytiotrophoblast cells,which are directly exposed to the maternal blood,is essential for the completion of transport and secretion of hormones.The microvilli of the syncytiotrophoblast cells,while,play a significant role in sensory stimulation and response.Many studies have shown that hyperglycemia or hyperinsulinemia have adverse effects on the structure and function of placenta,and but few have reported the effect of the combination of the two on the microvilli of the placenta.In this study,the morphological changes and potential mechanisms of microvilli of syncytiotrophoblast cells under different concentrations of glucose and insulin were investigated.Methods:(1)Transmission electron microscopy detected the microvillous morphology of syncytiotrophoblast cells in placental tissues of the normal pregnancy and GDM patients with full term pregnancy.(2)Scanning electron microscopy detected the microvillous morphology of HTR8-S/Vneo cells,in vitro,under fluidic culture for 24 hours by which the microvilli can be induced.Groups can be divided into:medium with normal concentration of glucose(5.5 mmol/L)or NG,medium with normal concentration of glucose(5.5 mmol/L)and low concentration of insulin(33.5mU/L)or NGLI,medium with normal concentration of glucose(5.5 mmol/L)and normal concentration of insulin(135mU/L)or NGNI,medium with high concentration of glucose(16.6 mmol/L)or HG,medium with high concentration of glucose(16.6 mmol/L)and low concentration of insulin(33.5mU/L)or HGLI,medium with high concentration of glucose(16.6 mmol/L)and normal concentration of insulin(135mU/L)or HGNI.The length and intensity of microvilli can be analyzed by statistical software of image pro plus.Of each group,length comes from the mean of length of 10 eyesight and intensity comes from the number divided by 25μm2 and mean of the value of 10 eyesight.(3)Western-bloting(WB),used for detecting the expression of protein ROCK2,phosphorylation modification level of PI3K,confilin,Akt and Ezrin.HTR8-S/Vneo cells were treated with different concentration of glucose or(and)insulin as above.(4)Laser confocal fluorescence microscopy(LCFM),used for detecting the uptaking for 2-NBDG,distribution of FITC-p-Ezrin and TRITC-F-actin.HTR8-S/Vneo cells were treated with different concentration of glucose or(and)insulin as above.Results:(1)Disordered,shortened and sparse arrangement of the microvilli can be found on trophoblast cells of placenta tissue belonged to the patients with GDM compared to normal puerperae.(2)For the length and intensity of microvilli of HTR8-S/Vneo cells,they are shortened(P<0.001)and sparse(P<0.01)which belonged to the cells which cultured with high concentration of glucose(HG)than normal(NG);Among cells of HG group,phosphorylation level of Akt and Ezrin decreased(P<0.01,P<0.01);intracellular diffusion of p-Ezrin increased;fluorescence intensity of 2-NBDG increased(P<0.001)and so did the F-actin label;phosphorylation level of PI3K p85 increased(P<0.001)but expression level of ROCK2 declined(P<0.05)and so declined the phosphorylation level of confilin(P<0.01).(3)For the length and intensity of microvilli of HTR8-S/Vneo cells,they are shortened(P<0.001)and sparse(P<0.001)which belonged to the cells cultured with high glucose and normal insulin(HGNI)than with normal concentration of glucose and insulin(NGNI),and no change on length but sparse(P<0.001)which belonged to the cells cultured with high glucose and low insulin(HGLI)than NGNI,and shorter(P<0.001)but no change on intensity which belonged to the cells cultured with normal glucose and low insulin(NGLI)than NGNI.(4)For the phosphorylation level of Akt and Ezrin of HTR8 cells,they decreased evidently(P<0.001,P<0.001)of HGNI than NGNI,and so decreased the diffusion of p-Ezrin;and decreased evidently(P<0.001,P<0.001)of HGLI than NGNI,and so decreased the diffusion of p-Ezrin;and decreased(P<0.05,P<0.05)of NGLI than NGNI,and so decreased the diffusion of p-Ezrin.(5)For the phosphorylation level of PI3K catalytic subunit p85 of HTR8 cells,it increased evidently(P<0.001)but with no change on PI3K p55 and decreased expression of ROCK2(P<0.01)and phosphorylation level of confilin(P<0.01)of HGNI than NGNI;and increased evidently(P<0.001)but with no change on PI3K p55 and decreased expression of ROCK2(P<0.05)and phosphorylation level of confilin(P<0.05)of HGLI than NGNI;and decreased evidently(P<0.001)but with no change on PI3K p55 and increased expression of ROCK2(P<0.01)and phosphorylation level of confilin(P<0.01)of NGLI than NGNI.Conclusion:The normal length and density of microvilli of HTR8-S/Vneo cells can be maintained cultured with normal concentration of glucose and insulin.Lower length and density can be observed with high concentration of glucose and insulin.Lower length but density can be observed with low concentration of insulin but glucose.Lower density but length can be observed with high concentration of glucose but low concentration of insulin.Influence for microvilli of HTR8-S/Vneo cells resulted by different concentration of glucose and insulin may completed by regulating the passway associated with Akt,PI3K and ROCK2 and then changing the expression of cytoskeleton associated protein like Ezrin and confilin.