| Objectives To investigate the effects of estrogen(17β- estradiol) on proliferation and the process of differentiating into bladder smooth muscle cells of adipose-derived stem cells(ASCs) in vitro.Methods Adipose-derived stem cells were isolated and cultured from female SD rats’ inguinal subcutaneous fat by collagenase digestion. Flow cytometry was used to determine the expression of the following surface antigens: CD90, CD73, CD45. Adipogenic differentiation and osteogenic differentiationthe were induced to determine the multi-directional differentiation potential. Adipose-derived stem cells were seeded at a density of 1,000 cells per well in 96-well plates and culture with DMEM contains different concentrations of estrogen(10-7M, 10-8M, 10-9M, 10-10 M, 10-11 M, 0). From day 1 to 8, cell proliferation assay was performed by MTS and the growth curves under the effect of each concentration of estrogen was drawn respectively. The most suitable concentration was chosen for investigating the effect on the process of differentiating into bladder smooth muscle cells, adipose-derived stem cells were induced by smooth muscle inductive medium with or without estrogen. Immunofluorescence, Real-time PCR, and Western Blot were used for the evaluation of the differences of the results between the two groups after 2 weeks and 4 weeks, smooth muscle-specific markers(α-SMA, SM22, Calponin, MHC) and estrogen receptor(ERα) were detected.Results Statistical analysis showed that estrogen promoted proliferation of adipose-drived stem cells and the concentration of 10-9mol/L is the most suitable concentration of estrogen. Western blot and real-time PCR revealed that after 2 weeks of differentiation into smooth muscle, the expression of all markers for smooth muscle cells in group of adipose-drived stem cells induced by SMIM with estrogen was significantly enhanced compare with the group without estrogen(p < 0.05). As the induction continued to 4 weeks, there was no significant difference in the expression of α-SMA, SM22 and Calponin between the two groups, while the expression of MHC in group of induced by SMIM with estrogen was significantly enhanced than the group of without estrogen(p < 0.05). In addition, the expression of estrogen receptor(ERα) in the group with estrogen increased compared with the group without estrogen(p < 0.05), and it is higher after 4 weeks than 2 weeks(p < 0.05).Conclusions(1) Estrogen promotes the proliferation of adipose-derived stem cells and the most suitable concentration is 10-9mol/L.(2) Estrogen enhances process of differentiating into bladder smooth muscle cells of adipose-derived stem cells in vitro by accelerating the maturation of ASCs derived smooth muscle cells.(3) The mechanism of the effects of estrogen proliferation and myogenic differentiation of adipose-derived stem cells fat stem cells may involve the activation of ERα. |
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