Effects of estradiol on proliferation and apoptosis in human amniotic mesenchymal stromal cells

Abstract:

Objective：To explore the effects of different concentrations of estradiol（E2） on proliferation and apoptosis in human amni－otic mesenchymal stromal cells（HAMSCs）. Methods：The improved enzyme digestion method was used to isolated culture HAMSCs. Flow cytometry analysis and immunofluorescence staining were used to identify the cultured cell. HAMSCs were divided into control group and E2 group（E2 with concentration of 10-5，10-6，10-7，10-8 mol/L were added respectively）. The proliferative ability of HAMSCs in each group was measured by cell counting Kit-8 assay（CCK-8）. Real-time PCR method was applied to measure the expression of the mRNA associated with pluripotency（Oct-4） and the expression of apoptosis-related genes Bcl-2 and Bax. The expression of apoptosis-related protein Bcl-2 and Bax was measured by Western blot. Results：CCK-8 showed that the proliferation activity of HAMSCs in each concentration E2 group and control group changed with time（Ftime=2418.132，Ptime=0.000）. There was no significant difference between the the proliferation rate of the E2 group of each concentration in the 1 to 4 days and the control group，while the proliferation rate of each concentration E2 group was signifi－cantly higher than that of the control group after 4 days（Fgroup=246.601，Pgroup=0.000），and there was no significant difference among the concentration groups. Real-time PCR analysis showed that mRNA expression of Oct-4 was significant higher in 10-6 mol/L E2 group（1.00±0.10） than in control group（0.58±0.16） and other E2 concentration group（1.31±0.22，0.81±0.04，0.78±0.07）（P=0.000，P=0.020，P=0.001，P=0.001）. In addition to 10-6 mol/L E2 group（0.83±0.47）（P=0.084），the expression of Bcl-2 mRNA was significant lower in other E2 concentration group（0.49±0.25，0.38±0.19，0.32±0.10） than in control group（1.06±0.13）（P=0.038，P=0.015，P=0.010），and mRNA expression of Bcl-2 was significant higher in 10-5 mol/L E2 group（2.20±0.58） than in control group（1.00±0.05） and other E2 concentration group（1.52±0.38，1.38±0.17，1.17±0.19）（P=0.001，P=0.028，P=0.012，P=0.003）. Western blot showed that protein expression of Bax was significant higher in 10-5 mol/L E2（0.514±0.014） group than control group（0.201±0.017）（P=0.000）. Nevertheless，protein expression of Bcl-2 was no significant higher in 10-5 mol/L E2 group（0.366±0.024） than in control group（0.215±0.080）（P=0.055）. Protein expression of Bax was significant lower in 10-6 mol/L E2 group（0.378±0.016） than in other E2 concentration group（0.514±0.014，0.547±0.036，0.577±0.028）（P=0.000）. Conclusions：The present study demonstrates E2 can promote the proliferation of HAMSCs，proliferative effect was time dependency. And high concentration of E2 group（10-5 mol/L） can promote apoptosis，however higher concentration of E2 group（10-6 mol/L） can maintain activity of HAMSCs and protect pluripotency.