Osteopontin Upregulate MMp9's Expression Through MAPK Signaling Pathway And Embryo Implantation Correlation Analysis

Objective: To investigate the chang rules of OPN (osteopontin) in the pregnant mouse's endometrial and confirm whether OPN can promotes the expression of MMP9 in human endometrial carcinoma cells RL95-2 cell (mimic endometrial cells during the implantation period ) through some pathway ,and infer OPN's infections and mechanism in the blastocysts implantation process.Methods: (1)Extract proteins and mRNA of mouse's endometrial cells in the first second third fourth fifth day of mouse's pregnant period , and use western blotting method and RT-PCR method to detect the OPN's expression . (2) Use OPN recombinant protein to stimulate RL95-2 cells , and detect whether MMP9's expression has changed , and whether some pathway was actived . (3) Transfect plasmid (with OPN variant A express gene and anti-Amp express gene) into RL95-2 cells , detect the expression changing of MMP9's protein and mRNA in RL95-2 cells and in cell medium supernatant .Results: (1) The results indicate that OPN's expression changed obviously in the mouse's endometrial tissues during pregnant period , and increase day by day during the pregnant time , and reach to the maximum at the fourth day (the mouse's blastocysts implanation day) ,and decrease after that day .. (2) After stimulating RL95-2 cells with OPN recombinant protein , MMP9's express quantity increase obviously , and the increase has dose dependent with the addition amount of OPN recombinant protein , and after stimulation , ERK1/2 was phosphorylated , and pass the MAPK signaling pathway the downstream molecule MMP9 was activated . OPN stimulation of ERK1/2 phosphorylation has time dependent , after stimulated for 1 hour, reached the peak. (3) The construction of plasmid(with OPN variant A express gene) was successful , but the transfection ratio was too low , the transfection method should be improved.Conclusions:(1)During pregnancy beginning to embryo implantation stage , OPN's expression presents increasing trend, and at the implantation window period the expression reach to the peak value, after that stage the expression of OPN declines。So OPN may participate in the process of blastocyst implantation.(2)During the blastocyst implantation process , OPN's participation mechanism is to phosphorylate ERK1/2 and activate MAPK pathway to promote the downstream molecule MMP9's expression , and hydrolyze endometrial epithelial cells to benefit blastocysts'implantation .(3)The construction of OPN's plasmid was successful.