Molecular Abnormalities Of Pulmonary Hypoplasia Of Congenital Diaphragmatic Hernia And The Study Of Its Mechanism

Objective:Congenital diaphragmatic hernia(CDH),a common congenital structural abnormality of the fetus,has a prevalence of about 1/3000.The main features of the disease are: lack of fetal diaphragmatic muscle during pregnancy resulting in abdominal organs herniation into the chest and caused pressing of the lung tissue.At present,the etiology of congenital diaphragmatic hernia is not clear,by the common impact of genes and the environment.With the continuous improvement of prenatal diagnostic techniques,fetal ultrasound during pregnancy can diagnose most congenital diaphragmatic hernia.Fetal MRI can be supplemented to confirm the diagnosis.CDH can be divided into mild and severe diaphragmatic hernia based on the observed-to-expected lung-to-head ratio,whether the liver is herniated into the thorax,whether or not other structural abnormalities are combined.The prognosis for children with mild CDH is relatively good,but in the long-term follow-up we found that most of the surviving patients still had long-term complications such as chronic infections and obstructive pulmonary disease.Severe congenital diaphragmatic hernia fetus mortality is higher,the current main treatment for foetal endoscopic tracheal occlusion(FETO),however,the development of foetal endoscopic facing a higher risk of premature rupture of membranes,premature birth.Therefore,the study of the treatment of congenital diaphragmatic hernia and the pathogenesis of pulmonary dysplasia is particularly important.Facing the development of maternal-fetal medicine and multidisciplinary cooperation,the treatment of congenital diaphragmatic hernia fetuses has also been gradually improved.Currently,a clear diagnosis of congenital diaphragmatic hernia fetus through close monitoring during pregnancy,improve the relevant check to rule out chromosomal abnormalities,through obstetrician and pediatrician,anesthesiologists and other related departments of multidisciplinary cooperation in the delivery period during ex utero intrapartum treatment(EXIT)is transferred directly to the Neonatal Intensive Care Unit for monitoring.Neonatal cardiopulmonary function is stable and can be surgically introduced to neonatal surgery for diaphragmatic repair followed by neonatal surgery.Neonatal respiratory management is the key to congenital diaphragmatic hernia treatment.Neonatal deaths are also mainly due to respiratory failure,which result from fetal lung hypoplasia.The current study shows that CDH fetal pulmonary hypoplasia is not only due to the abdominal cavity herniation into the chest cavity during the development and pressed lung tissue,another main reason is that CDH fetus throughout the lung development may be related to the various stages of regulation abnormalities which caused by abnormal molecules that controls lung development.Therefore,a clear congenital diaphragmatic hernia fetal pulmonary hypoplasia etiology to improve the survival of CDH neonates is particularly crucial.The advantage of whole exome sequencing(WES)in prenatal diagnosis of fetal abnormality has gradually been found.WES can detect abnormal changes in the gene level,so as to provide evidence to clarify the etiology and treatment of the disease.At present,the whole exon sequencing study of fetus with congenital diaphragmatic hernia is rare,and mainly for the study of foreign populations.We selected the pedigree of CDH fetus for the first time and conducted WES analysis.We found abnormal changes of lung development related genes in fetus with CDH.Ephrin/Eph signaling pathway,as a key factor in the regulation of vascular development,has a complex inter-regulation mechanism with other key molecules that regulate lung development such as VEGF,and has not been studied in the field of CDH,so we chose it as the target gene.Eph receptor family is the largest family of receptor tyrosine kinases.The Eph receptor family is divided into the Eph A and Eph B receptor subfamilies,the Eph A receptor subfamily anchored to the membrane by GPI junctions,and the transmembrane domain belonging to the Eph B receptor subfamily.Eph receptors and their ligands are highly expressed in the nervous system and in vascular endothelial cells.The receptors bind to the corresponding Ephrin ligands,respectively,to generate bi-directional signals that play roles in embryogenesis,axon guidance,synapse formation,and vascular development.The role of Ephrin B2 / Eph B4 in the development of the vascular system has been demonstrated by a knockout mouse model suggesting that Ephrin B2/Eph B4 is biologically functional through cell-cell interconnections and is essential for angiogenesis,gene knockout can cause mouse vascular system dysplasia,embryonic death.Studies by Salvucci et al.show that the effect of Eph B2 receptors and their transmembrane ligands on inducing angiogenesis in vitro is similar to that of Ang and VEGF.Therefore,we speculate that the process of lung branching may be regulated. To investigate the temporal and spatial expression changes of EFNB2 and EPHB4 in congenital diaphragmatic hernia rats and the mechanism of its regulation of lung development to provide a theoretical basis for the treatment of congenital diaphragmatic hernia during pregnancy in clinical.Method:1.Select the fetus with simple left congenital diaphragmatic hernia in Shengjing Hospital of China Medical University of 10 cases,collecting the cord blood and peripheral blood of fetuses' s parents for whole exome sequencing.Through data comparison,mutation identification and mutation screening deep analysis,congenital diaphragmatic hernia related mutation genes of 10 congenital diaphragmatic hernia fetuses were screened out.Consult literature related to lung development and screen for key genes that regulate lung development.Use STRING online tools for protein interaction analysis.We found that EFNB2 and EPHB4 are molecules that regulate lung development and are closely associated with the presence of abnormal VEGF and ANGPT1,so we hypothesize that it may play a key role in the regulation of lung development.2.Establishment of rat model of congenital diaphragmatic hernia and observation of fetal rat lung tissue abnormalities.The pregnant rats were randomly divided into two groups: experimental group(CDH group)and control group.At E8.5,females in the experimental group were treated with 100 mg of nitrofen(100mg dissolved in 1ml of oil),and females in the control group were treated with the same amount of oil.At 21.5 days of pregnancy,female mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution(40 mg/kg),the uterus of pregnant mice was dissected and the embryos were dissected.The defects of fetal rat diaphragm development were observed.The abdominal organs(liver hernia into the chest.Calculate the number of fetal rats with diaphragmatic defects and the total number of fetal rats.First of all,congenital diaphragmatic hernia fetal pulmonary hypoplasia general observation.Then take E21.5 days fetal rat lung tissue,sterile gauze to dry amniotic fluid and other tissues,weighed and recorded on an electronic balance,calculate the lung index.The mean alveolar number and mean linear intercept of the control group and the congenital diaphragmatic hernia group were calculated after HE staining on E21.5 day fetal rat lung tissue.E13.5 days,lung tissue culture in vitro,cultured for 96 h,observed and calculated two groups of lung tissue terminal bud number,area and perimeter.3.Take the E13.5,E15.5,E17.5,E19.5,E21.5 day fetal rat lung tissue,by Real-Time PCR,Western-Blotting,immunohistochemical detection of EFNB2 and EPHB4 at different developmental stages spatio-temporal expression changes and expression in lung tissue location.4.Take the E13.5,E15.5,E17.5,E19.5,E21.5 day fetal rat lung tissue,through the Western-Blotting technology to detect different lung tissue ERK phosphorylation level.E13.5 days fetal rat lung tissue culture,divided into CDH group and CDH + EFNB2 group,cultured for 96 h,calculate and compare the two groups of lung tissue terminal bud number,area and perimeter.The level of phosphorylation of ERK,P38,JNK and STAT3 in the two groups were detected by Western-Blotting after 96 hours of culture in vitro.Result:1.By STRING analysis,we found that IGF1 R,VEGFA,ANGPT1,MMP-2,EFNB2,WNT11,GLI3 and EPHB4 are genes closely related to congenital diaphragmatic hernia lung tissue development,and these complex molecules have a complex regulatory mechanism.According to the literature,we chose EFNB2 and its receptor EPHB4 as the research object.2.In this study,53 rats were given nitrofen to induced deformities,and 398 fetal rats of which 261 were malformed.Teratogenic rate was: 65.58%.In line with the literature teratogenic rate.Modeling successful.Congenital diaphragmatic hernia group deformity fetal rat lung tissue was significantly compressed,the volume is smaller than the normal lung tissue,and the lung index,the mean alveoli number and mean linear intercept was lower than the control group.E13.5 days fetal rat lung tissue culture in vitro at E13.5 days and after culture for 96 h,congenital diaphragmatic hernia group lung tissue development lagged behind in the control group,the number of lung terminal buds,perimeter and area were smaller than the control group.3.At E13.5 and E15.5 days(embryonic and pseudoglandular period of lung development),m RNA levels of EFNB2 and EPHB4 were not significantly different from those of the control group.With lung development,at E17.5,E19.5,E21.5 days(canalicular period and saccular period/ alveolar period of lung development)EFNB2 and EPHB4 compared with the control group showed a high expression trend.At E13.5 and E15.5 days(embryonic and pseudoglandular period of lung development),there was no significant difference in EFNB2 protein levels between the control group and the E17.5,E19.5,E21.5 days EFNB2 protein expression levels higher than the control group.For EPHB4,E13.5 days,there was no significant difference between the two groups;EPHB4 also showed high expression in congenital diaphragmatic hernia group as lung tissue continued to develop.3.In the study of the mechanism by which EFNB2 regulates the development of lung tissue,we found that the expression level of p-ERK in congenital diaphragmatic hernia group was lower than control group at E17.5 and 21.5 days.In vitro cultured E13.5 day lung tissue to 96 h,CDH + EFNB2 group lung tissue development was better than the control group,the performance of the terminal buds number increased,the area increased,the perimeter increased.We detected that the phosphorylation levels of ERK,P38,JNK and STAT3 were decreased in the lung tissue of exogenous EFNB2 recombinant protein group.Conclusion: 1.The mutations of lung development-related gene in congenital diaphragmatic hernia fetus may lead to pulmonary hypoplasia.2.EFNB2 / EPHB4 showed high expression in canalicular period and saccular period/ alveolar period of lung development in congenital diaphragmatic hernia fetal rats,and mainly expressed in epithelial cells.2.EFNB2 can promote the development of lung tissue of congenital diaphragmatic hernia fetal rats through MAPK and STAT3 signaling pathway.