Function Study Of LIF And β1, 4-galactosyltransferase Ⅰin Embryo Development And Implantation

Objective:Leukemia inhibitory factor(LIF) is named by inducing M1-leukemia cells differentiation into macrophage and inhibiting leukemia cells multiplication. LIF,which is a secretory glycoprotein with various biological functions, is the most important cytokine of embryo implantation. LIF plays a very important role in the embryo growth, development and differentiation. Recent evidence suggests that LIF culture medium could promote embry development, accelerate incubator, improve blastocyst survival rate, promote trophoblast cell proliferation,promote inner cell mass growth and accelerate zona pellucida removal.As It has been known that some implantation-related factors can be regulated by LIF, such as matrix metalloproteinases(MMPs),epidermal growth factor(EGF) et.al, but the mechanism of the role of LIF in controlling the expression ofβ1,4-GalTⅠis not clear.β1,4-GalTⅠis the earliest found glycosyltransferase and also the most intensively studied glycosyltransferase among the known glycosyltransferase family. It is found in two distinct subcellular structures, long style within the trans-Golgi compartment and short style on the plasma membrance. Functions in different manners. In the Golgi,β1,4-GalTⅠcatalyzes the transfer of galactose from UDP-Gal to terminal N- acetylglucoamine (GlcNAc) residues on glycoconjugates, forming the Galβ1→4 GlcNAc structer. Cell surfaceβ1,4-GalTⅠacts as a recognition molecule and participates in a number of cellular interaction,including adhesion and spreading of cell, recognition of sperm-ovum, growth of nerve neurite, immigration of mesenchymal and tumor, late morula compaction. Embryo implantation is similar to tumor metabasis and invasion. this thesis (or paper) contributes to the deep insight of the reaction between LIF andβ1,4-GalT and also the their functions in the on embryo development and implantation by using the method of RT-PCR, cytochemical staining and Dot-blot. The in vitro implantation model of monolayers of uterine epithelial (RL 95-2) cells and human trophoblast cell line(JAR) are used herein.Methods: LIF and LIFAb were added into human uterine endometrium cells (RL 95-2). RL 95-2 were analyzed by cytochemical staining, RT-PCR, Dot-blot and the in vitro modle which consisted of RL 95-2 and JAR. The effects of LIF and LIF-Ab treating cells onβ1,4-GalTⅠexpression and embryo imlantation were studied.Results: (1) The experimental results we obtain indicates that cytochemical staining results in an increased synthesis of GAlβ1-4GlcNAc in the treating LIF group, andβ1,4-GalTⅠgene,β1,4-GalTⅠexpression increased by RT-PCR and Dot-blot analyses in 24 hours after LIF was added into cells, both in comparison with the normal culture group. At the same time,the modle of embryo implantation in vitro showed the rate of embryo attachment of treating LIF group (83.5%) was higher than that of normal culture group(70%).But after treating cells with LIFAb for 24h,the synthesis of Galβ1-4GlcNAc became lower ,the expressive level ofβ1,4-GalTⅠgene was lower ,the synthesis ofβ1 , 4-GalTⅠprotein also became lower.Meanwhile,the rate of attachment was lower than the normal culture group(70%).( p<0.05)(2)The results showed green fluorescent protein (GPF) were expressed in cells after plasmids had been transfected into cells for 60 hours. Compared to normal culture group ,empty vector group and interference control group, the synthesis of Galβ1-4GlcNAc declined in interference group, but increased in overexpressed group. We have the observation that the overexpressed group,the expression of LIF gene and protein increased, the rate of embryo attachment and outgrowth(84.5%) was higher than normal culture group(70.0%), empty vector group (68.4%)and interference control group( 67.6%).(p<0.05)In interference group,β1,4-GalTⅠgene and expression declined and the rate of embryo attachment and outgrowth (29.2%) was also obviously lower.( p<0.05) Conclusion:(1) The results indicate there is interaction between LIF (RL95-2) andβ1,4-GalTⅠexpression.(2) The influence of LIF embryo implantation may be concerned with the expression of up-regulating endometria cells which isβ1,4-GalTⅠ.(3) Theβ1,4-GalTⅠexpression showed some relationship with embryo to embryo implantation. A possible approach can be built based on the results that the embryo identification and adhesion may be adjusted by regulating the LIF expression.The function ofβ1,4-GalTⅠin embryo implantation needs further investigation.