Objective: To investigate the method of establishing damaged endometrial stromal cells (ESC) model in vitro.
Methods: (1) From June to December 2011 ESC from normal endometrim at proliferation phase (n = 8) and secretory phase (n = 8) were isolated, cultured and identified in vitro. (2) ESC was treated with different concentrations of mifepristone or withdrawal of mifepristone at different time point. The proliferation inhibition percent was measured by cell counting kit-8 (CCK-8). (3) 0 µmol/L(control group)and 60 µmol/L (experimental group) concentration of mifepristone was added into ESC for 48 hours, then withdrew of mifepristone, continued to be cultured for 48 hours. The morphological changes were observed and apoptosis of ESC in different menstrual cycle were detected by flow cytometry. The mRNA and protein level of vascular endothelial growth factor (VEGF), caspase-3, 8, and 9 were determined by one-step quantitative real-time PCR (Q-PCR) and western blot.
Results: (1) ESC from 16 specimens of endometrium were all isolated and cultured successfully. (2) The proliferation inhibition rate of ESC was correlated with concentration and duration of mifepristone positively. The proliferation of ESC could be recovered at a range of time after withdrawal of mifepristone. However, when the concentration of mifepristone was 100 µmol/L, the growth of ESC recovered very hardly. (3) The damaged ESC spacing increased, the spindle shape and vacuolization in the cytoplasm were observed in experimental group; the rate of apoptosis of these damaged cells was significantly increased compared with control groups, which were (52 ± 12)% vs. (13 ± 5)% at the proliferative phase and (53 ± 6)% vs. (32 ± 3)% at the secretory phase (all P < 0.05). The relative mRNA level of VEGF was 0.52 ± 0.12 in experimental group and 1.00 ± 0.17 in control group at proliferation phase (P < 0.05). And the relative mRNA level of VEGF was 0.19 ± 0.03 in experimental group and 0.81 ± 0.07 in control group at secretory phase (P < 0.05). The relative level of VEGF protein in the experimental group were both decreased 1.98 and 2.79 folds at the proliferation phase and the secretory phase when compared with those in control group, respectively (P < 0.05). While the relative levels of caspase-3, 8, 9 mRNA were 5.62 ± 0.65, 5.41 ± 0.53, 7.22 ± 0.51 in the experimental group and 1.00 ± 0.44, 1.00 ± 0.21, 1.00 ± 0.32 in control group at the proliferative phase. In the mean time, the relative levels of caspase-3, 8, 9 mRNA were 10.22 ± 0.72, 25.3 ± 1.72, 9.48 ± 1.89 in experimental group and 1.42 ± 0.14, 1.14 ± 0.28, 1.16 ± 0.12 in control group at the secretory phase, respectively (P < 0.05). Compared with the control group, the levels of caspase protein in the experimental group were increased 2.04 and 1.60 folds in caspase-3, 4.23 and 1.49 folds in caspase-8, 2.65 and 3.5 folds in caspase-9 at the proliferative phase and at the secretory phase, respectively (P < 0.05).
Conclusion: The damaged model of ESC can be established after 48 hours by the withdrawal of 60 µmol/L mifepristone in treatment of ESC for 48 hours.