| BackgroundMicrovesicles(MV)are lipid bilayer membranous vesicles released by cells with diameters of 100-1000 nm.They selectively package a variety of bioactive substances from source cells.Such as lipids,cytokines,chemokines,growth factors,specific and non-specific proteins,DNA,m RNA,microRNA,lnc RNA,circ RNA,etc.[1]mediate the transmission of intercellular signals.Recent studies have shown that blastocysts can secrete microvesicles and act on recipient cells to alter their function and behavior[2].In mammals,the establishment of pregnancy requires pregnancy recognition signals from the embryo,followed by embryo implantation and placenta formation[3].Embryo blastocyst is the result of the"dialogue"with maternal endometrium,blastocysts in proto-oncogenes,adhesion molecules such as orderly under the control of maternal endometrial invasion,in this process,the endometrium in a variety of cytokines,estrogen,progesterone and adhesion factor of the network system,under the precise control of receptive of embryos.During this period,the endometrial epithelial cells undergo significant morphological and structural changes to adapt to embryo implantation.Its main characteristic is epithelial mesenchymal transformation(EMT),that is,epithelial cells first lose polarity and intercellular adhesion,then gain invasion and migration ability,and finally transform into mesenchymal phenotype[4].Communication between cells has traditionally been thought of as either direct cell-to-cell contact or the transmission of information through signaling molecules secreted by cells.Recent studies have shown that the intercellular transfer of MV is also an important cellular communication mode[5],and the messenger function of microvesicles is mainly dependent on the phenotype of the source cell[6].Therefore,we hypothesized that blastocyst microvesicles are involved in the dialogue between blastocyst and endometrial cells,and may be related to endometrial epithelial-mesenchymal transformation.Therefore,in this study,blastocysts and primary endometrium cells of 3.5 days of gestation BABL/C mice were used as materials to establish a co-culture system of blastocyst microvesicles and endometrium cells,and to analyze the effect of pre-implantation blastocyst MV on the epithelial-mesenchymal transformation of endometrium cells.To verify the possibility that blastocyst microvesicles can induce the transformation of endometrial epithelial cells into mesenchymal cells,and to provide a basis for the MV communication during embryo implantation.Object1.To establish an optimized method for primary isolation and purification of mouse endometrial epithelial cells.2.To investigate the phenomenon and possible mechanism of epithelial-mesenchymal transformation induced by mouse blastocyst microvesicles.Methods1.In the two methods,epithelial cells were separated into clusters and adhered to the wall slowly.5×105 cells were inoculated in a 24-well plate,and after 24h,the cells could grow to 80%slippage.Neither of them could be cultured by trypsin digestion.Morphology and Cytokeratin-8 specific antibody of epithelial cells identified as epithelial cells.Flow cytometry was used to identify the purity of the epithelial cells after Dispase ? and trypsin digestion(Method A),and the purity of the epithelial cells after collagenase I and DNase digestion(Method B)was 60%.Method A could simply and efficiently culture primary epithelial cells with biological characteristics of epithelial cells.It is a good method for primary isolation and purification of mouse endometrial epithelial cells.2.MV was separated and purified by ultra-high speed centrifugation combined with density gradient centrifugation.The morphology was observed by transmission electron microscopy,the size distribution was measured by nano particle tracker,and the expression of TSG101 and CD63 was analyzed by WB.A co-culture model of blastocyst MV and endometrium epithelial cells(mEEC)was established.The dynamic changes of the invasion power of endometrium epithelial cells were monitored by ECIS in real time within 120 hours.Taking 12 h,36 h,48 h,60 h,84 h and 120 h as time nodes,the resistance values of pre-implantation blastocyst MV and mEE cell co-culture group and mEE cell control group were compared and analyzed.The composition of m V miRNAs was analyzed by q PCR.Through bioinformatics analysis of these miRNAs and their target genes,the miRNAs that might promote the invasion of recipient cells were predicted.3.PMKH26 was used to stain and trace blastocyst MV,and the co-culture system of pre-implantation blastocyst MV and mEE cells was established.The groups of 0 h,48h and 96 h were set,respectively.The process of MV entering mEE cells before implantation was detected by laser confocal microscopy.The mEE cell group was used as the control group.The influence of pre-implantation blastoblastoblast MV on the expression of E-cadherin and Vimentin in mEE cells in co-culture system was detected by Western-blot and cellular immunohistochemistry.The expression of E-cadherin and Vimentin in cells was quantitatively analyzed by Image J software.Results1.In the two methods,epithelial cells were separated into clusters and adhered to the wall slowly.5×105 cells were inoculated in a 24-well plate,and after 24 h,the cells could grow to 80%slippage.Neither of them could be cultured by trypsin digestion.Morphology and Cytokeratin-8 specific antibody of epithelial cells identified as epithelial cells.The purity of epithelial cells extracted by flow cytometry method A was80%and the purity of epithelial cells extracted by method B was 60%.Method A could simply and efficiently culture primary epithelial cells with biological characteristics of epithelial cells,which was A good method for primary isolation and purification of mouse endometrial epithelial cells.2.After 3.5 days of gestation,a kind of spherical microvesicle with a diameter of about 200 nm was isolated by ultra-high speed centrifugation combined with density gradient centrifugation.The microvesicle characteristic proteins TSG101 and CD63were expressed.ECIS results showed that the invasion of mEE cells and mouse blastocysts at 12 h,24 h,36 h and was not significantly different from that of mEE cells control group(P<0.05),When cultured for 48 h and 60 h,the invasion of mEE cells in the experimental group was significantly higher than that in the control group(P<0.05),the invasion power of mEE cells in the experimental group was significantly higher than that in the control group at 84 h and 120 h(P<0.01).PCR results showed that there were 224 invasion-related miRNAs in pre-implantation blastocyst MV,accounting for nearly half of the total invasion-related miRNAs,and 80 miRNAs could promote invasion.Although the number of miRNAs that inhibit invasion is dominant,some miRNAs that promote invasion have been expressed at a high level in MV,such as miR-346 and miR-21.It is suggested that EMT induced by MV in mouse blastocysts may be regulated by the PI3K-Akt pathway activated by MV carrying miRNA-21targeting PTEN.3.Pre-implantation blastocyst MV was co-cultured with mEE cells for 0 h.No p KH26 labeled MV was found in mEE cells.After co-culture for 48 h,a small amount of Pre-implantation blastocyst MV was collected in the cytoplasm of mEE cells.Compared with co-culture for 0h,the uptake rate of p KH26-labeled MV of mEE cells cultured for 48 h and 96 h was significantly higher(P<0.01).Meanwhile,the expression of E-caderhin in mEE cells was significantly down-regulated(P<0.05,P<0.01),the expression of Vimentin in mEE cells was significantly up-regulated(P<0.05,P<0.01).Conclusions1.The primary endometrial epithelial cells were obtained by digestion of uterine tissue with DISPase ? enzyme and trypsin and purification by differential adhesion,which was a simple and efficient method for the primary separation and purification of mouse endometrial epithelial cells.2.The co-culture system of pre-implantation blastocyst MV and mEE cells confirmed that pre-implantation blastocyst MV could promote the invasion ability of mEE cells,and could affect the expression of E-caderhin and Vimentin in mEE cells.Based on the types and characteristics of miRNAs contained in mouse blastocyst MV,these results suggest that the possibility of enhanced invasion of mEE cells to transform into mesenchymal cells is related to the PI3K-Akt pathway activation by miRNA-21targeting PTEN in mouse blastocyst. |
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