| PART I ISOLATION OF MOUSE ENDOMETRIAL STROMAL CELLS AND IN VITRO DECIDUALIZATIONObjective:To isolate mouse primary endometrial stromal cells and induce in vitro decidualization by estrogen and progesterone.Methods:Uterus were isolated from mice on day 4 of pregnancy and cut into pieces. The pieces were incubated in a mix of dispase and collagenase Type I. The supernatant was allowed to stand for 2-3 minutes and then passed through a 200-mesh stainless steel sieve. Cells were plated and incubated for 1 h. The unattached cells were removed by several washes. Cells were identified by immunofluorescence base on that vimentin staining was positive in stromal cells but not in the epithelial cells, and cytokeratin is positive in the epithelial cells. Cells in model group were treated with estrogen and progesterone to induce in vitro decidualization. Cells in control group were treated without estrogen and progesterone. Expression of decidual prolactin-related protein (dPRP) mRNA was monitored by quantitative polymerase chain reaction (qPCR).Results:Immunofluorescence analysis showed that the ratio of endometrial stromal cells to total cells was 95.6±2.9%. The relative expression of dPRP mRNA in model group and control group was 1.00000±0.00000 and 0.00017± 0.00004, respectively.Conclusion:Mouse endometrial stromal cells can be successfully isolated and induced in vitro decidualization.PART Ⅱ EFFECTS OF DIFFERENT CONCENTRATIONS OF DEHYDROEPIANDROSTERONE ON PROLIFERATION AND DPRP MRNA EXPRESSION IN MOUSE ENDOMETRIAL DECIDUALIZING CELLSObjective:To investigate the effects of different concentrations of dehydroepiandrosterone (DHEA) on proliferation and decidual prolactin-related protein (dPRP) mRNA expression in mouse endometrial decidualizing cells.Methods:Primary endometrial stromal cells were isolated and cultured in vitro from mice on day 4 of pregnancy. The cells were divided into experimental group 1, group 2, group 3, group 4, model group and control group. The experimental groups and the model group were treated with estradiol and progesterone to induce in vitro decidualization. Experimental groups were exposed to 0.1,1,10,50μmol/L DHEA, respectively. The control group was not treated with estradiol, progesterone and DHEA. Cultured for 72 h, expression of decidual prolactin-related protein (dPRP) mRNA was monitored by RT-PCR. Morphological changes were observed under inverted microscope after 24 and 48 h. The cells of experimental groups and model group were cultured in 96-well plates. Cell proliferation measure was performed using a Cell Counting Kit-8 after 72h.Results:The relative expression of dPRP mRNA in experimental group 1,2,3,4 was 0.978±0.258,0.764±0.135,0.250±0.022,0.013±0.000, respectively. The model group was 1.000±0.000. The expression of dPRP mRNA in both group 3 and 4 was lower than model group, P<0.05. Group 2 compared with group 4, P< 0.05. There is no statistical significance between other groups. Cells in control group showed a fibroblast-like appearance. Cells in model group enlarged and had an increased amount of cytoplasm. Appearance of cells in group 1 and 2 was similar to model group. Cells in group 3 and 4 became polygonal with less cytoplasm. The absorbance of samples at 460 nm in group 1-4 was 1.2228±0.0382,0.7905±0.2071,0.5925±0.2298,0.2658± 0.0374, respectively. The absorbance of model group was 1.1497±0.1062. P <0.05 when group 3 and 4 compared with the model group. P<0.05 when group 2,3,4 compared with the group 1, respectively; P> 0.05 when group 2 compared with group 3, while P<0.05 when compared with group 4; P> 0.05 when group 3 compared with group 4.Conclusion:The proliferation and dPRP mRNA expression of mouse endometrial stromal cells is not significantly affected when exposed to 0.1 and 1 μmol/L DHEA, while 10 and 50μmol/L DHEA inhibits cell proliferation, and down-regulates dPRP mRNA expression. |
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