Expression And Function Of CXCL12,CXCR4 And CXCR7 In Morbidity Adherent Placenta

Placenta accreta is associated with increased maternal morbidity and mortality.A characteristic feature of placenta accreta is over-invasion of trophoblasts with the abnormally dilated vessels.However,the mechanism underlying over-invasion phenomenon of placenta accreta remains unclear.Because of the CXCL12,CXCR4 and CXCR7 with actions of adhesion and angiogenesis,we therefore hypothesized those chemokines may play important roles in placenta accreta.To test the question,we examined CXCL12,CXCR4 and CXCR7 expressions in placenta tissue sections of placenta accreta including implanted sites and non-implanted sites compared with normal placentation.We further investigated circulating CXCL12 levels in women with the pathologic diagnosis of the third trimester placenta accreta.In vitro,the changes of proliferation,migration,invasiveness,cell apoptosis were observed followed by over expression and RNA interfence of CXCL12,CXCR4 and CXCR7 in HTR-8/SVneo and JAR cell lines to explore their potential role and mechanisms.Part 1 Expression of CXCL12,CXCR4 and CXCR7 in Placental Trophoblastics of Placenta AccretaObjectives This study was aimed to detect the expressions of CXCL12,CXCR4 and CXCR7 in placental tissues of third trimester placenta accteta and to explore the relationship between CXCL12,CXCR4 and CXCR7 and over-invasion of trophoblasts.Methods The study was a case-control designed at the First Affiliated Hospital of Guangxi Medical University from 2010 to 2015.Thirty-nine cases of placenta accrete in this part were included,17 accreta,16 increta and 6 percreta.Thirty-nine health pregnancies who underwent cesarean section for previous cesarean deliveries were matched for gestational age and served as controls.CXCL12,CXCR4,and CXCR7 immunolocalization in placental tissues were analyzed.Clinical data were obtained from each subject’s medical records.Results 1)The frequency of cesarean hysterectomy section in placenta accreta group was significantly higher than that in normal control.2)CXCL12and receptor CXCR4 and CXCR7 protein were mainly expressed in placental trophoblastic cells.3)Scores of CXCL12,CXCR4 and CXCR7 between implanted and non-implanted sites from the same case of placenta accreta were not different(CXCL12:P=0.724;CXCR4:P=0.609;CXCR7:P=0.745).Scores of CXCL12,CXCR4 and CXCR7 in implanted site were significantly higher than those in controls(CXCL12:P<0.001;CXCR4 为:P<0.001;CXCR7:P <0.001).4)There was a significant trend toward a higher median staining scores of CXCL12,CXCR4 and CXCR7 by depth of invasion(accreta,increta,percreta)(P<0.001).Conclusion The higher expressions of CXCL12,CXCR4 and CXCR7 in placental trophoblasts of placenta accreta are related to the depth oftrophoblastic invasion.CXCL12,CXCR4 and CXCR7 may play an important role in the development of placenta accreta.Part 2 Levels of CXCL12 in Maternal Blood of Women with Placenta AccretaObjectives This study was aimed to detect the level of CXCL12 in circulating blood and placental trophoblastic cells of third trimester placenta accreta and to explore whether CXCL12 could be a biochemical mark for prediction of placenta accreta.Methods The study was a case-control design at 3 tertiary referral hospitals of Nanning between January 2016 and September 2017.33 cases of placenta accrete were included,10 percreta,14 increta,and 9 accreta.33 placenta previa and 33 normal controls were matched at sampling for gestational age to placenta accrete.CXCL12 levels were investigated by ELISA in blood and immunolocalization in placental tissue.Clinical data were obtained from each subject’s medical records.Results 1)Maternal plasma CXCL12 was increased in placenta accrete[3144.19(SD 701.56)pg/m L] compared to placenta previa[2249.02(SD 501.97)pg/m L](P < 0.001)and normal controls [2207.42 SD(441.84)pg/m L](P <0.001).2)When cases were further detailed classified the different depths,plasma CXCL12 levels among placenta percreta[3476.09(SD 499.73)pg/m L],increta [3046.89(SD 643.40)pg/m L],accrete [2926.76(SD 898.69)pg/m L]were no significant difference(P=0.188).3)CXCL12 levels in blood of placenta accrete were correlated with staining scores of trophoblasts(r=0.789,P<0.001).4)ROC curve showed a cutoff value of plasma CXCL12 leve1(2581.12pg/m L)with the sensitivity of 87.9% and the specificity of 78.8% for the discrimination of pathologically diagnosed placenta accrete.The AUC was 0.879(95% CI:0.794-0.964).Conclusion Because of a positive correlation of CXCL12 levels in placental tissue and maternal blood,it is reasonable to consider that placental trophoblasts might be one significant source.It potentially offers some form of non-radiological test to determine whether there is significant adherence of the placenta.Part 3 Effect of CXCL12,CXCR4 and CXCR7 Proteins on Biological Function of Trophoblast Cell LinesObjectives The aim of this study was to investigate the effect of CXCL12,CXCR4 and CXCR7 on biological function of human extravillous trophoblast cell line HTR-8/SVneo and chorioepithelioma cell line JAR.Methods 1)Lentiviral vectors in that CXCL12,CXCR4 and CXCR7 genes or interfering mi RNAs of CXCL12,CXCR4 and CXCR7 were inserted were transferred into HTR-8/SVneo and JAR cells.After antibiotic resistant screening,CXCL12,CXCR4 and CXCR7 stably silenced or overexpressed HTR-8/SVneo and JAR cell lines were established.2)The tansfection efficiency was observed though fluorescence microscope.The levels of silence or overexpression CXCL12,CXCR4 and CXCR7 in HTR-8/SVneo and JAR cells transfected were detected by real time quantitative PCR and Western blot analysis.3)Effect of CXCL12,CXCR4 and CXCR7 on the cell proliferation,migration,invasiveness,cell apoptosis in HTR-8/SVneo and JAR cell lines were investigated by CCK-8,wound healing,transwell assay and flow cytometric analysis.Results 1)CXCL12,CXCR4 and CXCR7 stably silenced or overexpressed HTR-8/SVneo and JAR cell lines were established.2)The down regulation of CXCL12,CXCR4 and CXCR7 genes in HTR-8/SVneo and JAR cells inhibited the ability of cells proliferation,migration and invasion by CCK-8 test,wound healing and transwell assay,while up-regulation of CXCL12,CXCR4 and CXCR7 enhanced proliferation,migration and invasion.3)Over expression or up-regulation of CXCL12,CXCR4 and CXCR7 did not change cells apoptosis.Conclusion In vitro test showed that CXCL12,CXCR4 and CXCR7 can affect the biological function of trophoblast cell lines,such as proliferation,migration and invasiveness.Therefore,CXCL12,CXCR4 and CXCR7 play important roles in over-invasion of trophoblasts in placenta accreta.Part 4 Effects of regulated CXCL12,CXCR4 and CXCR7 Expressions on Trophoblasts InvasivenessObjectives The aim of this study was to explore the possible mechanism that was regulation of CXCL12,CXCR4 and CXCR7 on the invasion of trophoblast cells.Methods 1)Expression of Rho A,Rac-1 and Cdc42 were detected by Western blot.2)Expression and phosphorylation of the proteins in Rho/Rock,PI3K/AKT signaling pathways were detected by western blot.3)In CXCL12,CXCR4 and CXCR7 stably overexpressed cells,the Rock or PI3 K inhibitors were added,then cells of invasion were detected by transwell invasion assay.Results 1)Down-regulated CXCL12,CXCR4 and CXCR7 in HTR-8/SVneo and JAR cellscan obviously restain the expression levels of Rho A,Rac-1 and Cdc42.Mean while the phosphorylation of MLC and Akt proteins were suppressed.up-regulated CXCL12,CXCR4 and CXCR7 promoted the expression levels of Rho A,Rac-1 and Cdc42,mean while the phosphorylation of MLC and Akt proteins,too.2)Using Rock or Akt inhibitors to block Rho/Rock or PI3K/AKT signaling pathways,the ability of cells invasion was reduced by Transwell assay.Conclusion Regulation of CXCL12,CXCR4 and CXCR7 in HTR-8/SVneo and JAR cell lines can affect the cells invasiveness,which is through Rho/Rock and PI3K/AKT pathways and change the expression levels of Rho A,Rac-1 and Cdc42.