Detection And Analysis Of Abnormal Expression Of Notch3 Affecting The Differentiation Of Trophoblast And Its Regulatory Mechanism

Syncytiotrophoblast cell layer is an important part of the maternal-fetal barrier,involved in human placental transport and hormone production which are required to the fetoplacental development.Notch signal is a key pathway in participating in determining cell fate and maintaining adults tissue homeostasis during development.Notch3 is a key factor of Notch signal pathway also which might control trophoblast cells fate in placenta,but its role in human syncytiotrophoblast fusion,and whether its abnormal regulation is involved in the occurrence of preeclampsia,is yet known clearly.Herein,we used human early villous,term placenta,human primary trophoblast,and BeWo cells fusion models to verify the expression of Notch3 in human placental trophoblast,and to clarify the role of Notch3 in the process of trophoblast syncytialization.We arrested BeWo cells cycle by using serum-free starvation in culture to verify the effect of cell cycle arrest on the trophoblast syncytialization,and utilized hypoxia culturing BeWo cells model to verify the effect of hypoxia on the trophoblast syncytialization.Ad-N3ICD adenovirus was injected through tail vein in pregnant mice to set up a Notch3 forced expression model,to clarify the impact of Notch3 on pregnant mice and to explore the role of Notch3 in the pathogenesis of preeclampsia.The current research results are as follows:1.The Notch3 expression in the human placental trophoblast.Notch3 expression was detected in human primary trophoblast syncytialization model,human villous during early pregnancy and syncytiotrophoblast renewal villous tissues,human term placenta,by qRT-PCR,immunohistochemistry and immunofluorescence.The results indicated that Notch3 and other Notch family receptors and ligands mRNA levels were down-regulated in human primary trophoblast syncytialization model;Notch3 was mainly expressed in mononuclear cytotrophoblast cells and expressed weakly in syncytiotrophoblast cells in human first trimester pregnancy villous,syncytiotrophoblast renewal villous tissues and the term placenta.2.The role of Notch3 in the BeWo cells fusion.BeWo cells fusion were induced by forskolin?FSK?,and DAPT,a?-secretase inhibitor,was used to prevent Notch3 signal activation by inhibiting Notch3 intracellular domain?N3ICD?transfer into the nucleus.We explored the role of Notch3 on BeWo cells fusion.The results showed that Notch3 expression was down-regulated during the fusion of BeWo cells.Following DAPT treatment,were treated with,the level of N3ICD in the BeWo cell's nucleus was decreased and the Notch3 signal activation was suppressed,and the fusion ratio was enhanced.3.The effect of cell cycle arrest on the fusion of BeWo cells.Serum-free starvation was induced in the BeWo cell cycle arrest at G₁phase.Immunofluorescence,qRT-PCR,Western blot,and flow cytometry were used to determine the effect of BeWo cells cycle arrest on the fusion process.The results indicated that the cell cycle arrested at G₁ phase,had significantly higher fusion ration in BeWo cells as compared with controls.4.The effect of hypoxia on the fusion of BeWo cells.The BeWo cells exposed to hypoxia were used to study the how Notch3signal pathway regulated the fusion of BeWo cells.The results showed that under 2%O₂ hypoxia,the expression of Notch3 was increased,and the ability of BeWo cells fusion was impaired;the nuclear protein components of BeWo cells were detected by Western blot,and N3ICD in the nucleus of BeWo cells was increased.Besides,sFLT1 was detected in BeWo cell culture medium by ELISA,and the result showed that hypoxia increased the secretion of sFLT1 protein during BeWo cells fusion.5.Preeclampsia-like features in Notch3 overexpressed pregnant mice.Ad-N3ICD adenovirus was used to construct a Notch3 overexpression mouse model during pregnancy.Adenovirus was injected through the tail vein on the 6th day of pregnancy?GD6?.The blood pressure,urinary protein,serum sFLT1 level and fetal weight in pregnant mice were measured to clarify the effect of Notch3 on pregnancy outcomes in pregnant mice and to explore the effect of Notch3 on preeclampsia.The results indicated that after injection of Ad-N3ICD on GD6,the mRNA level of N3ICD in the placenta of pregnant mice was increased significantly,and Notch3 overexpressing pregnant mice were successfully constructed.The blood pressure,urinary protein,serum sFLT1 levels were up-regulated after injection of Ad-N3ICD,and the fetal weight were decreased at GD18.These results showed preeclampsia-like features in those mice.Based on the above results,this study suggested that Notch3 expression was down-regulated during trophoblast syncytialization;hypoxia increased the expression of Notch3 and promoted N3ICD transfer into nucleus to regulated the cell cycle progression,affecting the self-renewal and differentiation balance of trophoblast cells,thereby inhibiting trophoblast syncytialization and may participate in the development of preeclampsia.