Effects Of Notch Pathway On Biological Characteristics Of Trophoblast Cells In Sheep

Embryo implantation is a process in which animal blastocyst trophoblast cells and maternal endometrial epithelial cells gradually establish tissue and physiological connections.In this process,the trophoblast cells develop from the embryonic trophoblast cells before the implantation is completed into the placental trophoblast cells after the implantation.The Notch pathway can regulate cell fate decision during development and play an important role in regulating cell proliferation,differentiation and apoptosis,migration and invasion,and is closely related to the occurrence and development of various cells,tissues and organs,and plays an important role in tissue and embryonic development.Notch signaling is activated by ligand-receptor interactions between adjacent cells,promotesγ-secretion-dependent cleavage of Notch receptors,and releases the Notch intracellular domain（NICD）into the nucleus,where NICD interacts with transcription factors CSL binds to activate this pathway,thereby regulating cell differentiation,invasion,adhesion and other processes.The Notch pathway can regulate the process of ovine embryo engraftment by regulating the biological function of ovine trophoblast cells.Objective:To study the effects of Notch pathway activator JAG-1 and Notch pathway inhibitor DAPT on the proliferation,apoptosis,cycle and migration of ovine trophoblast cells;Method:1.In this study,trophoblast cells were isolated and cultured by tissue block culture method,trypsin digestion method and trypsin and tissue block method,cells were purified by trypsinization.Observation of morphological characteristics and growth characteristics of trophoblast cells under microscope.The specific expression of cytokeratin 7（CK7）in trophoblast cells was identified by immunocytochemical staining,and the marker gene interferon-τof sheep trophoblast cells was detected by RT-PCR.Trophoblasts were treated with progesterone（P₄）,estradiol（E₂）or progesterone（P₄）,estradiol（E₂）and interferon-tau（IFN-τ）simultaneously,and the transcription levels of embryonic implantation-related genes were detected by real-time quantitative PCR（qRT-PCR）.2.Sheep trophoblast cells were cultured in vitro,and the trophoblast cells were divided into Notch pathway activation group,Notch pathway inhibition group and blank control group,Notch pathway activation group and Notch pathway inhibitor group.The effects of JAG-1 and DAPT on cell proliferation were detected by CCK8 method;PCR and WB methods were used to detect the transcription and protein expression levels of Notch1 in each group of cells.q RT-PCR was used to detect the Notch pathway downstream gene Jagged-1,hairy division-related enhancer 1（Hes1）,vascular endothelial growth factor and q RT-PCR in each group of cells.（VEGF）transcription level,WB method to detect the expression levels of apoptosis-related proteins Caspase-9,Bcl-2,Bax,Cyt c,qRT-PCR to detect the m RNA expression of cell cycle-related genes CCNA2,CCNB1,Cyclin D1.3.Cell scratch test was used to detect the migration of cells in each group.q RT-PCR was used to detect the transcription levels of invasion-related genes MMP-2 and MMP-9 in each group of cells.ELISA and q RT-PCR were used to detect the effect of Notch pathway on prostaglandin E₂and F_2αprostatic secretion and prostaglandin synthase expression.Result:1.The results showed that among the three cell culture methods,the use of trypsin and tissue block method had a shorter cell growth cycle,the cells migrated from the tissue block on the 2nd day,and the cells spread out in a single layer on the 5th to 6th day,and the morphology of the cells was observed under the microscope and showed epithelioid growth with different cell shapes.The cells were identified by immunocytochemical staining,and the results showed that the cells were positive for CK7,and the positive rate was greater than 95%.RT-PCR detected that the isolated sheep trophoblast cells could express IFN-τnormally.In addition,after treatment with progesterone（P₄）,estradiol（E₂）and interferon-τ（IFN-τ）,real-time quantitative PCR results showed that the expression levels of embryo implantation-related genes ISG15,CXCL10,RSAD2,CTSL,SPP1,and MUC1 were up-regulated.It was confirmed that the cells could reflect the main characteristics of the maternal placental trophoblast in the process of embryo implantation,and provided a trophoblast cell basis for subsequent experiments.2.CCK8 results showed that DAPT significantly inhibited trophoblast cell proliferation in a time-and dose-dependent manner,while JAG-1 had no effect on trophoblast cell viability.The q RT-PCR results showed that compared to the control group,the expression levels of Jagged-1,Hes1 and VEGF were significantly upregulated in the activation group（P<0.01）,while the expression levels of Notch1,Jagged-1,Hes1 and VEGF in the inhibition group were significantly decreased（P<0.01）,the transcript levels of CCNA2 and CCNB1 in the activation and inhibition groups were significantly decreased,while the expression level of cyclin D1 was significantly increased in the activation group and significantly increased in the inhibition group reduced.The WB results showed that compared to the control group,the Notch1protein activation group was significantly decreased and the inhibition group was significantly increased;Apoptosis-related proteins,expression of the apoptosis-inhibiting protein Bcl-2 decreased（P<0.05）in the inhibition group,and expression of the pro-apoptotic protein Bax,Caspase9,Cyt c increased（P<0.05）,and the expression of apoptosis-related proteins in the activation group was the opposite.It indicated that the Notch pathway affects the proliferation and apoptosis of ovine trophoblast cells.3.and the activator group increased the expression of apoptosis-related proteins to the opposite.q RT-PCR results showed that compared with the control group,the expression levels of MMP-2 and MMP-9 in the activator group were significantly up-regulated（P<0.01）,while the expression levels of MMP-9 and VEGF in the inhibitor group were significantly increased（P<0.05）.MMP-2 was not statistically significant.ELISA results showed that compared with the control group,PGE₂in the activator group was significantly increased,and PGF_2αin the inhibitor group was significantly decreased,and there was no significant difference in PGE₂between the inhibitor group and the activator group（P>0.05）.The results of q RT-PCR showed that compared with the control group,the expressions of PTGS1 and PGFS were significantly decreased in the inhibitor group,and the expressions of PTGS1 and PTGES were significantly increased.significantly reduced.It indicated that the Notch pathway affects the migration and invasion ability of ovine trophoblast cells.Conclusion:In this study,the primary trophoblast cells of Kazakh sheep were successfully isolated and purified by trypsin and tissue block culture method combined with trypsin differential time digestion method.The decrease of Notch1 expression can inhibit the proliferation of trophoblast cells and promote cell apoptosis.The viability of trophoblast cells was not affected and apoptosis was reduced;activation of the Notch pathway promoted the migration and invasion of trophoblast cells,while inhibition of the Notch pathway reduced the migration and invasion ability of trophoblast cells in vitro.It shows that the Notch pathway regulates the basic functions of ovine trophoblast cells,and lays a foundation for further research on the effect of Notch pathway on sheep embryo engraftment.