| Objective: Neural tube defects(NTDs)is one of the common congenital malformations,the mechanism of which is still unclear.Some infants can survive by surgical treatment,but urine,fecal incontinence and lower limb dysfunction are common complications that require lifelong treatment,seriously affecting the improvement of quality of life.Therefore,it is of great significance to explore a new diagnosis method of NTDs for early interventions and treatments.At present,the prenatal diagnosis of NTDs mainly relies on imaging examination,but due to its requirement for high diagnostic level and expensive equipment,it can only be carried out in large prenatal diagnosis centers.At the same time,imaging can only give a clear diagnosis after the occurrence of severe structural abnormalities,which cannot meet the requirements of early diagnosis and treatment.Maternal peripheral blood molecular markers are expected to be used for noninvasive diagnosis before the occurrence of significant structural abnormalities.However,existing diagnostic markers,such as alpha-fetoprotein(AFP),lack good diagnostic specificity despite their significance in the extensive screening of NTDs.It is of great significance to search for earlier and more specific diagnostic markers for current clinical diagnosis.Now,proteomics is a relatively mature technique for finding new molecular biomarkers.In previous studies in NTDs,we found some potential diagnostic molecular biomarkers in spinal cord,amniotic fluid and serum for neural tube defects.In recent years,with the rapid development of researches in the area of exosomes,it has been found that exosomes may be a significant way for fetal-derived molecules to enter maternal peripheral circulation,and it will be more possible to find molecular indicators specific to abnormalities by screening molecular biomarkers in exosomes.Therefore,in this study,proteomics was utilized to analyze proteins with differential expression in exosomes and tried to find specific protein markers for neural NTDs.In addition,the previous study of our research group found that skeletal protein LMNA had a downregulated expression in the maternal serum of fetuses NTDs and congenital heart disease(CHD),and believed that it could be used as a new diagnostic biomarker.However,fetal-derived skeletal protein LMNA is not a secreted protein,and the mechanism by which it traverses placenta barrier into maternal circulation is unclear.Therefore,the cell model of overexpressing LMNA-GFP fusion protein was used in this study to explore the pathway and mechanism of its entry into maternal circulation.Methods: In the first part of this study,we used retinoic acid-induced NTDs Wistar rat model to perform proteomic detection of serum exosomes between normal and NTDs rats at pregnant day E18 and combined with bioinformatics analysis to search for differentially expressed proteins potentially involved in multiple biological processes.In the subsequent validation,we expanded the sample size to verify the variation trends of the screened protein biomarkers by Western blot and ELISA,and detected earlier serum exosomes from E12 to E18 days.Western blot and immunohistochemistry(IHC)were used to study its expression in spinal cord tissue.In the second part of this study,we used c57 mouse model of CHD induced by nitrofen to conduct Western blot and immunohistochemical analysis of LMNA in serum,serum exosomes,amniotic fluid,placenta and heart tissues of E17 pregnant mice from normal group and malformation group.Then,a plasmid expressing LMNA-GFP fusion protein was constructed and transfected into amniotic fluid mesenchymal stem cells.The distribution of GFPlabeled LMNA in maternal tissues was observed by amniotic cavity injection of transfected cells and exosomes secreted by transfected cells,and the pathway and mechanism of LMNA entering maternal circulation were discussed.Results: 1.Three proteins that related to each other in the same protein interaction network were found through serum exosome proteomics combined with bioinformatics analysis in pregnant mice: ACTR2,CORO1 A and DNM2.All these proteins are involved in the polymeric remodeling of f-actin,which is closely related to nerve development,neural tube closure,and vesicle formation.2.Western blot and ELISA were used to verify the expression of ACTR2,CORO1 A and DNM2 in the serum exosomes of pregnant rats with NTDs from E12 to E18 days,and the results were consistent with that of serum exosome proteomics.At the same time,the expression of ACTR2,CORO1 A and DNM2 in the serum was studied,and it was found that there was no significant change in the whole serum,but only had significant down-regulation trends in the serum exosomes.3.Western blot and IHC were used to study the expression of ACTR2,CORO1 A and DNM2 in spinal cord tissues,which proved that the variation trends of these three proteins in tissues was consistent with that in blood exosomes,and their specific expression in spinal cord tissues and nerve cells was found,especially the expression of CORO1 A and DNM2 was more specific,which were also down-regulated in fetal neural exosomes.4.ELISA was used to detect the expression change of CORO1 A and DNM2 in the serum exosomes of pregnant women,which proved that they were also downregulated in the serum exosomes of pregnant women with NTDS fetuses at 12-40 weeks of gestation,and the expression change was more obvious at early gestation(12-18 weeks),with better diagnostic effect.5.Western blot was used to verify that the LMNA protein in the C57 mouse model of CHD induced by nitrofen showed a down-regulated change trend in the serum of pregnant mice,and the expression of LMNA was also down-regulated in the heart,amniotic fluid and placenta of fetal mice,which was also confirmed by the immunohistochemical results.The expression of LMNA in amniotic fluid exosomes was also found.6.The cell model expressing LMNA-GFP fusion protein was successfully constructed,and the existence of LMNA-GFP fusion protein was verified by Western blot and microscopic observation.7.Amniotic fluid mesenchymal stem cells expressing LMNA-GFP fusion protein and their exosomes were injected into the amniotic cavity respectively.Fluorescent markers on the fetal side were found under fluorescent microscopy,and Western blot detection of GFP labels proved that LMNA could enter the maternal blood circulation through cell migration and exosome.Conclusions: 1.CORO1 A and DNM2 in serum exosomes of pregnant women can potentially be used as novel molecular biomarkers for clinical application of the early non-invasive diagnosis of NTDs.2.Downregulated expression of ACTR2,CORO1 A and DNM2 in spinal cord was associated with NTDs.3.LMNA can enter the maternal circulation from the fetal side through cell migration and exosome. |
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