Study On The Relationship Between PLAC1 And Gestational Diabetes Mellitus

Type 1,type 2 diabetes and Gestational diabetes mellitus(GDM)are prevalent worldwide,with prevalence increasing over the past 20 years,especially in China.China has become one of the highest incidence of GDM in the world.GDM is a state of glucose intolerant and is found during pregnancy for the first time.It brings near-term complications,including the macrosomia,polyhydramnios,preeclampsia,neonatal hypoglycemia,neonatal hyperinsulinemia,followed by shoulder dystocia,Cesarean section,birth injury,postpartum hemorrhage and other risks to the mother,fetus and newborns.postpartum and offspring of GDM have a significant increase in the risk of type 2 diabetes mellitus(T2DM)in the future,and the incidence is younger.The risk of developing into GMD of the female offspring is significantly increased.It has a serious negative impact on the quality of our nation.Therefore,how to contain GDM from the source,is our primary research purpose.At present,GDM's etiology is still unknown.In recent years,studies show that the level of pregnancy hormone levels,autoimmune,genetic,adipokines levels,inflammatory factors levels are more or less aggravated the insulin resistance of GDM patients.But all the above-mentioned pathogenic factors can not fully explain the pathogenesis of GDM and the role of near-term adverse effects of the fetus and therefore GDM can not be predicted and prevention.The placenta is a temporary,but extremely important organ,which is produced by embryo during pregnancy.It is the only communication channel between the mother and the fetus.Maternal with a healthy physiological environment,in order to promote the placenta normal growth,development,maturity,so as to ensure its normal exercise of metabolic functions,complete the transport of nutrients and gases and protect the fetus to escape from mother's immune system to ensure the growth and development of the fetus.placenta is also a secretory organ,its synthesis of various types of protein and rich hormone feedback on the mother,causing the mother to local or systemic changes in order to maintain the smooth progress of pregnancy.Morphology of GDM placenta is larger,crisp and fragile.Under the microscope,50-60%of the villi is immature,villus diameter expansion,edema,stenosis of small vessel lumen,increased syncytiotrophoblast nodes,increased fibrin-like necrosis of the chorionic villi.At the same time,the ultrastructure of syncytiotrophoblast cells change significantly:the villi brushes of the free surface of the cells are short,sparse,mitochondrial swelling,rough endoplasmic reticulum expansion,and basement membrane bending thickening.Even if glycemia of GDM women are well controlled during pregnancy,placental morphology and histological changes can not be improved.Therefore,study of placental pathophysiology and its expression of genes,synthetic protein is the starting point for the control of GDM from the source.PLAC1(Placenta-specific 1)is expressed primarily on trophoblast cells of the placenta.PLAC1 protein contains 212 amino acids,molecular weight of about 28-30KDa,it has a highly conserved signal sequence,four o-glycosylation sites and 13 potential phosphorylation sites,suggesting it plays a key role in regulation of glucose metabolism and maintenance of normal Pregnancy.We hypothesized that changes in PL AC 1 expression cause abnormal energy metabolism,leading to the occurrence and/or development of GDM.The expression and the position of PLAC1 protein in the placenta.And correlation between PLAC1 and GDM pregnant women's clinical features was analyzed.At the same time,the effect of PLAC1 protein on trophoblast's function was investigated at the cell level.The cells were cultured hyperglycemia in order to explore the relationship between PLAC1,Leptin and insulin resistance of GDM.So as to further study the pathogenesis of GDM,to provide a basis for screening meaningful biological indicators,in order to predict the occurrence and development of GDM,improve the outcome of the mother and child.Part IExpression of PLAC1 in pregnant women with gestational diabetes mellitus and its potential clinical significanceObjectiveTo observe the expression and localization of PLAC1 in placenta of gestational diabetes mellitus(GDM),and to analyze the correlation between PLAC1 and GDM pregnant women's clinical features.Subjects and MethodsA total of seventy-five pregnant women with or without GDM were randomly recruited.37 pregnant women were diagnosed as GDM and 38 cases were as normal controls.The clinical data and information were collected,and the fasting blood samples and placental tissue of pregnant women were collected.The fasting blood glucose(FPG)was measured by glucose oxidase colorimetry.The levels of glycosylated hemoglobin(HbA1c),total cholesterol(TC),triglyceride(TG),high density lipoprotein(HDL)and low density lipoprotein(LDL)were measured by chemiluminescence and enzyme assay.Fasting insulin(Fins)was determined by enzyme-linked immunosorbent assay(ELISA).The expression and distribution of PLAC1 in placenta were measured by Western blot and immunohistochemistry.And the correlation between PLAC1 expression and clinical data and blood index was analyzed.Results1.The FPG(3.98±0.98mmol/L),HbA1c(5.44±0.41%),OGTT Oh(5.29±1.11 mmol/L),OGTT 1h(10.84±2.58mmol/L),OGTT 2h(8.89±1.72mmol/L)and the birth weight of the neonates(3606.76±445.06g)of GDM was significantly higher than that of the controls(3.58±0.65 mmol/L,5.08±0.60%,4.48±0.26 mmol/L,8.19±1.17 mmol/L,6.62±0.91 mmol/L,3404.47±337.52g,respectively),(p<0.05)?2.The expression of PLAC1 in placenta of GDM(1.08 ± 0.64)was lower than that of controls(1.75±1.28),and there was significant difference(p<0.01).3.Under normal pregnancy circumstances,PLAC1 protein was positively correlated with BMI when delivery,Fins,TG and HOMA-IR,but there was no correlation with these indicators while GDM occurred.4.Placental tissue immunohistochemistry examination results:GDM pregnant women placenta,immature villi,increased number of cell trophoblast cells,stenosis of small vessel lumen,increased syncytiotrophoblast nodes,increased fibrin-like necrosis of the villi.PLAC1 protein was strongly positive in brown tissue in placental tissue,and the expression was mainly distributed in the cytoplasm of villus interstitial and syncytiotrophoblast.The expression level of PLAC1 protein in placenta and maternal surface of control group was significantly higher than that of GDM group.ConclusionThe expression of PLAC1 protein in the cytoplasma of the trophoblast cells and the glial mesenchymal cells was significantly decreased in the placenta of GDM.Under normal pregnancy circumstances,PLAC1 protein was positively correlated with BMI when delivery,Fins,TG and HOMA-IR,but there was no correlation with these indicators while GDM occurred,which indicated that PLAC1 was closely related to the regulation of metabolic activity in vivo and prompted the occurrence and development of GDM.Part ?The effects of PLAC1 on biological behaviors in trophoblastObjectiveTo clarify the role of target gene PLAC1 in the regulation of trophoblast proliferation,migration,invasion.Subjects and MethodsThe expression of PLAC1 was down-regulated by PLAC1 small interfering sequence in trophoblast cell lines SWAN and JAR,and the expression of PLAC1 was up-regulated by PLAC1 plasmid in the above two cell lines.The proliferation of trophoblast cells was examined by CCK-8,and Transwell combined with or without matrigel matrix gel to detect trophoblast invasion and migration.Western blot was used to detect PLAC1 protein level.Results1.SWAN cell interference efficiency was 62%,while JAR cell was 58%.The upregulation efficiency of SWAN cells was 159%,while JAR cells was 278%.All had significant differences.2.In the trophoblast cell lines,down-regulation of PLAC1 expression can significantly inhibit the proliferation,invasion and migration of SWAN and JAR cells.3.In the trophoblast cell line,up-regulation of PLAC1 expression can significantly promote the proliferation,invasion and migration of SWAN and JAR cells.ConclusionThe lower expression of PLAC1 can significantly inhibit the proliferation,invasion and migration of trophoblast cells,suggesting that the decrease of PLAC1 expression can cause the dysfunction of trophoblast,and affect the normal growth and development of placenta which further affects the occurrence and development of maternal GDM and other gestational complications.Part IIIChanges of PLAC1 in trophoblast cells and its correlation with leptinObjectiveThrough the high glucose culture of trophoblasts to simulate the glucose changes in the body,to study the changes of PLAC1 under different glucose levels.And to understand the relationship between PLAC1 and leptin.Subjects and MethodsTrophoblast cell lines SWAN and JAR cells were cultured in medium with two different sugar concentrations and mannitol hypertonic medium.The expression of PLAC1 was down-regulated by PLAC1 small interfering sequence in trophoblast cell lines SWAN and JAR to observe the changes of leptin level in the above two kinds of cells and cell culture supernatant.The expression of PLAC1 was up-regulated by plasmid to observe the expression of leptin in the above two kinds of cells.The levels of PLAC1 and leptin protein were detected by Western blot.ELISA was used to detect leptin protein levels in cell culture supernatant.Results1.The expression of PLAC1 in trophoblast cells was significantly decreased.Meanwhile,the expression of Leptin and leptin secreted into culture supernatant were significantly increased.2.In the trophoblast cell lines,the synthesis and secretion of leptin was increased significantly after PLAC1 expression was down-regulated.3.In the trophoblast cell line,the expression of Leptin was significantly decreased after PLAC1 expression was up-regulated.ConclusionThe expression of PLAC1 decreased by the stimulated of high glucose.The decrease of PL AC 1 level could lead to the increase of leptin expression,while increase of PLAC1 level,could lead to the decrease of Leptin synthesis and secretion.The regulation of Leptin by PLAC1 may lead to the development of GDM.