Study On The Role Of HIF-1α/VEGF In The Pathogenesis Of Missed Abortion In Early Pregnancy And Its Related Regulatory Mechanism Under Hypoxia

Part I: Study on the relationship between Hi F-1α regulates of VEGF and the onset of missed abortionObjective: To detect the expression hypoxia-inducible factor-1α(Hypoxi-inducibl factor 1αlfa,Hi F-1α),vascular endothelial growth factor(vascular endothelial)in chorionic villi from patients with missed abortion,and the related functional tests of HTR8/svneo cells in the early hypoxic environment constructed in vitro,to show that the role of HIF-1α regulates VEGF in the formation of chorionic Villi and expounds its clinical significance in the pathogenesis of missed abortion.Method:1.Collect chorionic villi from 30 cases of missed abortion patients and30 cases of elective induce abortion in early pregnancy,using immunohistochemical method to detect the expressions of HIF-1α,VEGF and CD34 markers of Microvessel density(MVD)of the two groups.To further study the relationship between expression of HIF-1α,VEGF and MVD in villus tissue.2.To compare the HTR8/svneo cell proliferation,migration,invasion and the ability of and tube formation of two different oxygen environments after cultured 6,12,24,and 48 hours in vitro cultured under hypoxia(2%O2)and normal oxygen(20%O2).3.Western blot test the changes of HIF-1α protein after 6,12,24,and 48 hours of hypoxia and normal oxygen conditions.Test the expression of VEGF protein after 6,12,24,and 48 hours hypoxia culture using WB,RT-QPCR detect the changes of HIF-1α m RNA levels in hypoxia after 6,12,24,and 48 hours cultured.4.Study the change of target gene VEGF and cell tube-forming ability in si RNA technique silence HIF-1α.Results : 1.The microvessel density of villi in missed abortion group was significantly lower than that in early pregnancy(p<0.001);The average light density of HIF-1α and VEGF in missed abortion group was lower than that in early pregnancy(p<0.001).In early pregnancy,MVD was positively correlated with the expression of HIF-1α(r=0.644,p<0.001),and MVD was positively correlated with VEGF expression(r=0.695,p<0.001);In the missed abortion group,MVD was positively correlated with the expression of HIF-1α(r= 0.898,p<0.001),MVD and VEGF were positively correlated(r=0.940,p<0.001).2.There was no statistically significant difference in the proliferation,migration,invasion and the ability of and tube formation of HTR8/svneo cells between hypoxia and nomoxia conditions(p>0.05)after 6hours,there was a statistically significant difference in culture with 12-24-48hours(p<0.05),hypoxia was associated with the proliferation,migration,invasion and the ability of and tube formation of HTR8/svneo cells.3.HIF-1α protein expression was very rare under normoxia,the expression of HIF-1α protein was significantly higher under hypoxia,and the change of HIF-1α protein begain to increase in hypoxia after 12 hours of hypoxic treatment,and could stabilize to 48 hours.The changes of VEGF protein in hypoxia culture also showed time dependence,similar to the expression of HIF-1α protein in hypoxia.Hypoxia can stimulate HIF-1α m RNA transcription level after 12 hours of hypoxia stimulation,and can maintain to 24 hours,hypoxia stimulate HIF-1α m RNA transcription level consistent to protein level change in hpoxia.4.After silent HIF-1α with si RNA,the expression of VEGF protein was inhibited obviously,and the ability of tube formation of HTR8/svneo cells become decreased markedly.Conclusion:The incidence of missed abortion is related to the formation of chorionic villi approved from the clinical level and in vitro cell experiment.It was proved that Hif-1α regulates VEGF is the key factor of the formation of chorionic villi,and the downward adjustment of Hif-1α leads to the low expression of VEGF,which causes the formation of villi and causes the incidence of missed miscarriage.Part II: Study on the regulation of HIF-1Α/VEGF by PI3k/akt pathway under hypoxia and its relation to missed abortionObjective: To detect the expression of PI3K/ akt pathway key protein factor in missed abortion chorionic villus specimen and its cell-related experiment in hypoxia environment confirmed the effect of Pi3k/akt pathway on HIF-1Α/VEGF in hypoxia environment and the influence on cell tube-forming ability,and clarified pi3k/akt pathway regulation hif-1α/ Significance of VEGF in the pathogenesis of missed abortion.Method:1.The expression of PI3 K and AKT in two groups of chorionic Villi were determined by immunohistochemistry and the relationship with MVD was detected.2.The change of the level of HIF-1α,PI3 K,AKT and VEGF protein was measured by WB experiment after the effect of hypoxia condition,PI3 K inhibitor and si RNA technology silence HIF-1α.3.Detection of the cell’s ability to tube formationwith hypoxia cultured,PI3 K inhibitor cultured,si RNA technology silence HIF-1α.Results: 1.The average light density of PI3 K in missed abortion group was lower than that in early pregnancy(p=0.019).The average light density of akt in missed abortion group was lower than that in early pregnancy(p=0.046).In early pregnancy abortion group,MVD was positive correlation with PI3K(r=0.583,p=0.001),and was positively correlated with Akt(r=0.533,p=0.002).MVD was not positively correlated with PI3 K in the missed abortion group(r=0.264,p=0.159),and also was not positively correlated with Akt(r= 0.218,p=0.247).2.HIF-1α and VEGF activity is enhanced in HTR8/svneo cells in the hypoxia environment,simple PI3 K inhibitor action can reduce HIF-1α and VEGF in hypoxia environment.The expression level of akt under hypoxia is not decreased after Hif-1α silence.The activity of HIF-1α and VEGF was the lowest when the PI3 K inhibitor and HIF-1αwere silent.3.The tube formation of HTR8/svneo cells were the strongest in the low oxygen environment alone,and the PI3 K inhibitor could reduce the ability of tube formation of HTR8/svneo cells.In hypoxia environment,the PI3 K inhibitor combined with HIF-1αsilencing has the lowest capacity of tube formation of HTR8/svneo cellConclusion:The Pi3k/akt pathway activates Hif-1α and increases the expression of VEGF in hypoxia environment,which regulates the capacity of trophoblast cells.The low expression of pi3k/akt leads to the downward adjustment of HIF-1Α/VEGF,which may causes the formation of villi and the pathogenesis of missed abortion.Part III: The study of EGFR regulation HIF-1α/VEGF under hypoxia environment and its relation to missed abortionObjective: To detest the expression of EGFR in the missed abortion chorionic villus specimen,and the cell experiment in the low oxygen environment,confirmed the role of EGFR regulates HIF-1α/VEGF in hypoxia environment and the effect of cells tube formation,to show the ability to EGFR regulate HIF-1α/VEGF of in the pathogenesis of missed abortion.Methods: 1.The expression of EGFR and the relationship with MVD in two groups of Villi were determined by immunohistochemistry.2.The changes of EGFR,HIF-1α and VEGF protein levels were detected by WB experiment after the effect of hypoxia condition,EGFR inhibitor and si RNA technology silence HIF-1α.3.Detection of the cell’s ability to tube formationwith hypoxia cultured,EGFR inhibitor cultured,siRNA technology silence HIF-1α.Results: 1.The average light density of EGFR in missed abortion group was lower than that in early pregnancy(p=0.019).In early pregnancy,MVD was positively correlated with EGFR(r=0.581,p<0.01),and MVD was positively correlated with EGFR in missed abortion group(r=0.707,p<0.01).2.HTR8/svneo cells have the strongest HIF-1α/VEGF activity in a simple hypoxia environment, and EGFR inhibitors can reduce the activity of HIF-1α and VEGF under hypoxia.The expression level of EGFR under hypoxia is not decreased after Hif-1α silence.In hypoxia environment,EGFR inhibitor combined with HIF-1αsilencing have the lowest HIF-1Α/VEGF activity.3.The tube formation of HTR8/svneo cells are the strongest in the low oxygen environment alone,and EGFR inhibitors can reduce the tube formation ability of HTR8/svneo cells.In hypoxia environment,EGFR inhibitor combined with HIF-1α silencing have the lowest tube formation capacity of HTR8/svneo cell.Conclusion:The EGFR pathway activates Hif-1α and increases the expression of VEGF in hypoxia environment,which regulates the capacity of trophoblast cells.The low expression of EGFR leads to the downward adjustment of HIF-1Α/VEGF,which may causes the formation of villi and the pathogenesis of missed abortion.