Function Study Of IL-1,β1, 4-galactosyltransferase Ⅰ In Embryo Implantation And The Correlation Of Them On Other Implantation Factors Expression

Objective: The embryo implantation of mammalian animal is a complicated process. The successful implantation of the blastocyst depends on adequate interactions between the embryo and the uterus. The development of the embryo begins with the fertilized ovum, a single totipo- tent cell which undergoes mitosis and gives rise to a multicellular structure named blastocyst. At the same time, increasing concentrations of ovarian steroid hormones initiate a complex signaling cascade that stimulates the differentiation of endometrial stromal cells to decidual cells, preparing the uterus to lodge the embryo.Various factors changing with embryo implanta- tion progress promote the mutual recognition and adhesion of uterus- embryo.Interleukin-1(IL-1) is produced by macrophages, stromal cells and trophoblast cells, it is one of the most important cytokines of embryo implantation. IL-1 contains IL-1α,IL-1βand IL-1ra. Recent evidence suggests that in human endometrium IL-1αand IL-1βhave a ubiquitous presence in epithelium, stroma and endothelial cells. Successful implanta- tion of fertilized ovum has been correlated to high concentrations of both IL-1αand IL-1βin the surrounding environment of human embryos. Im- paired fertility was observed in IL-1R I knockout mice.β1,4-GalTⅠis one of the first glycosyltransferases to be purified and also the most intensively studied glycosyltransferase among the known glycosyltransferase family. It is localized in the trans-Golgi compartment of most eukaryotic cells, where it participates in the elongation of oligosac- charide chains on glycoproteins and glycolipids.β1,4-GalTⅠhas also been reported in the cell surface,where it has been suggested to function as a receptor for extracellular glycoside substrates.It has foundβ1,4-GalTⅠas a recognition molecule participated in the adhesion and spreading of cell, recognition of sperm-ovum, growth of nerve neurite, the formation of tissue and tumor metabasis as an adhesive molecule. Embryo implantation is similar to tumor metabasis and invasion.The paper studied the effects of IL-1 andβ1,4-GalTⅠon embryo and implantation. The correlation of IL-1 andβ1,4-GalTⅠon the other implantation factors expression was studied to investigate mutual relation of the factors and deepen the mechanism of embryo implantation.Methods:β1,4-GalTⅠgene interference plasmids,overexpression plasmids, interference control plasmids and empty vector plasmids were transfected into RL95-2 cells (to simulate endometrium during implanta- tion).To investigate the correlation ofβ1,4-GalTⅠon IL-1βand MMP-9 expression use RT-PCR and Dot-blot; IL-1β/IL-1ra were added into cell culture fluid of RL95-2 cells to co-culture, to observe the expression ofβ1,4-GalTⅠ,MMP-9 and COX-2 use RT-PCR; To research the change of the rate of embryo attachment use the in vitro implantation model of mono- layers of RL95-2 cells (to simulate endometrium during implantation) and JAR cells (to simulate embryo during implantation).Results:The results showed green fluorescent protein (GPF) were expressed in RL95-2 after plasmids had been transfected into RL95-2 for 60 hours; Compared to normal culture group, empty vector group and inter- ference control group, the expression ofβ1,4-GalTⅠdeclined in inter- ference group and increased in overexpressed group.These results indicated plasmid transfection was successful. MMP-9 gene expression and protein synthesis declined in interference group, and the rate of JAR cells attach- ment(36.2%) was also lower than that of normal culture group(72.1%), empty vector group (70.6%)and interference control group(68.7%). In over- expression group, MMP-9 gene expression and protein synthesis increased and the rate of JAR cells attachment(83.9%)was also higher. However,both in interference control group and overexpression group, IL-1β?gene ex- pression haven't significant differences. β?1,4-GalTⅠ,MMP-9 and COX-2 gene expression increased after IL-1βand RL95-2 cells have co-cultured for 24 hours;β?1,4-GalTⅠ,MMP-9 and COX-2 gene expression declined after IL-1ra had been add into cell culture fluid for 24 hours.Conclusion:These results indicatedβ1,4-GalTⅠmay participate in mutual adhesion of uterus-embryo and embryo implantation course by regulating MMP-9 expression.These results hinted IL-1βmay up-regulateβ1,4-GalTⅠ,MMP-9,COX-2 expression in the process of embryo implantation.