The Mechanism Of Improving Endometrial Receptivity In G-CSF

Objective: The purpose of this study was to observe the effect of G-CSF on the proliferation of human endometrial cells and to study its mechanism of action by culturing human endometrial cells in vitro and applying different concentrations of G-CSF to the cells.Methodology:1 The primary human endometrial epithelial cells and stromal cells were isolated and cultured in vitro by collagenase I digestion and centrifugation,and the morphology and structure of human endometrial cells were observed using an optical microscope and inverted phase contrast microscopy;Human endometrial epithelial cells and stromal cells were identified using immunocytochemical methods;2 The optimal concentration and duration were selected as the follow-up experimental groups after observing the effect of different G-CSF concentrations(0,50,100,200,500,800,and 1000 pg/mL)and durations(24 hours,48 hours,and 72 hours)acted on epithelial cells and stromal cells using MTT method;3 G-CSF receptors in cells were identified using immunofluorescence and immunocytochemistry methods;4 The mRNA expression levels of PCNA and SLP mRNA in the experimental group and the control group were detected using Real-time quantitative(RT)-PCR,and the differences between the two groups were compared;5 The expression levels of ?-catenin protein in the experimental group and the control group were detected using Western-blot,and the differences between the two groups was compared.Result:1.The success rate of separation of endometrial cells after digestion with 0.2% collagenase I and centrifugation was greater than 90%.After immunocytochemical staining,cytoplasm of epithelial cells was positive to keratin antibody,and cytoplasm of stromal cells was positive to vimentin antibody.2.MTT experiments showed that the cell survival rate was the highest for human endometrial epithelial cells being cultured in vitro for 48 hours at a G-CSF concentration >500 pg/ml.The cell survival rate was the highest for endometrial stromal cells being cultured in vitro at a G-CSF concentration of 500 pg/ml for 72 hours.3.Detection of G-CSF receptor by immunofluorescence revealed that the cytoplasm of epithelial cells was positive-red fluorescence,and the cytoplasm of stromal cells was positive-green fluorescence.Immunocytochemistry showed that the cytoplasm of epithelial cells and stromal cells was brownish yellow and the nucleus was negative with no coloration.The G-CSF receptor was found in the cytoplasm of epithelial and stromal cells.4.RT-PCR detection indicated that the relative mRNA expression of PCNA and SLP in the epithelial cells and stromal cells in the experimental group was increased compared with the control group.5.Western-blot detection indicated that the relative expression of ?-catenin protein in epithelial cells and stromal cells of the experimental group was increased compared with the control group.Conclusion:1.Human endometrial epithelial cells are with the highest cell viability when being cultured in vitro at a concentration of G-CSF >500 pg/ml for 48 hours.Endometrial stromal cells are with the highest cell viability when being cultured in vitro at a concentration of 500 pg/ml G-CSF for 72 hours.2.Endometrial epithelial cells and stromal cells exist G-CSF receptor,G-CSF is probably by promoting epithelial cells and stromal cells in the expression of PCNA gene and SLP,raise the Wnt signaling pathway in ?-catenin expression thus promotes endothelial cell proliferation.