Abstract:

Objective To investigate the effects of bisphenol A （BPA） on the proliferation activity and stemness characteristics of endometrial mesenchymal stem/stromal cells （eMSCs）， and to elucidate the improvement effect of human umbilical cord mesenchymal stem cell-derived supernatant （hUCMSC-Sup） on the cell injury. Methods The eMSCs were cultured in vitro and treated with different concentrations of BPA （0， 200， 250， 300， 350， and 400 μmol·L^-1）. The eMSCs were divided into control group（only cultured with culture solution）， BPA group （cultured with isovolumetric culture solution including 200 μmol·L^-1 BPA）， BPA+hUCMSC-Sup group （cultured with isovolumetric culture solution including 200 μmol·L^-1 BPA and 50% volumetric ratio of hUCMSC-Sup）， and BPA+CHIR-99021 group （cultured with isovolumetric culture solution including 200 μmol·L^-1 BPA and 10 μmol·L^-1 CHIR-99021）.The survival rates of eMSCs in various groups were detected by methyl thiazolyl tetrazolium（MTT） assay. The numbers and diameters of the spheroids in various groups were detected by spheroids formation assay， the proliferation activities of the cells in eMSCs stem cell spheroids in various groups were detected by CCK-8 assay； the percentage of CD73+ cells in eMSCs in various groups were detected by flow cytometry； the expression levels of sex determining region Y-box 2 （Sox2）， octamer-binding transcription factor 4（Oct4）， and Nanog mRNA in the eMSCs in various groups were detected by real-time fluorescence quantitative PCR（RT-qPCR） method， the expression levels of β-catenin protein in the eMSCs in various groups were detected by Western blotting method. Results The MTT results showed that after treated with BPA for 24 and 48 h， compared with 0 μmol·L^-1 BPA group， the survival rates of eMSCs in 200， 250， 300， 350，and 400 μmol·L^-1 BPA groups were significantly decreased （P<0.01）. At 24 and 48 h after treatment， compared with control group， the survival rate of the eMSCs in BPA group was significantly decreased （P<0.01）； at 48 h after treatment， compared with BPA group， the survival rate of the eMSCs in BPA+hUCMSC-Sup group was significantly inereased （P<0.05）. The spheroids formation assay results showed that compared with culture 3 d group， the numbers and diameters of stem cell spheroids of the eMSCs in culture 4 d group and culture 5 d group were significantly increased （P<0.05 or P<0.01）； compared with control group， after 48 h of culture， the number and diameter of the cells in eMSCs stem cell spheroids in BPA group were significantly decreased （P<0.05 or P<0.01）. The CCK-8 results showed that after 24 and 48 h of treatment， compared with control group， the proliferation activity of the cells in eMSCs stem cell spheroids in BPA group was significantly decreased （P<0.01）； compared with BPA group， the proliferation activity of the cells in eMSCs stem cell spheroids in BPA+hUCMSC-Sup group was significantly increased（P<0.01）. The flow cytometry results showed that compared with control group， the percentage of the CD73+ cells in the eMSCs in BPA group was significantly decreased （P<0.01）； compared with BPA group， the percentage of the CD73+ cells in eMSCs in BPA+hUCMSC-Sup group was significantly increased （P<0.01）. The RT-qPCR results showed that compared with control group， the expression levels of Sox2， Oct4， and Nanog mRNA in the cells in BPA group were significantly decreased （P<0.01）； compared with BPA group， the expression levels of Sox2， Oct4， and Nanog mRNA in the cells in BPA+hUCMSC-Sup group and BPA+CHIR-99021 group were significantly increased （P<0.01）. The Western blotting results showed that compared with control group， the expression level of β-catenin protein in the eMSCs in BPA group was significantly decreased（P<0.01）； compared with BPA group， the expression levels of β-catenin protein in the eMSCs in BPA+hUCMSC-Sup group and BPA+CHIR-99021 group were signifrcantly inereased （P<0.01）. Conclusion BPA can inhibit the stemness characteristics of the eMSCs， and injury the self-renewal and repair of endometrium； its mechanism may be related to down-regulating the activity of Wnt/β-catenin signal pathway in the cells. hUCMSC-Sup can promote the proliferation of injured eMSCs， and has improvement effect on the stemness injury induced by BPA.

Key words: Bisphenol A, Endometrial mesenchymal stem/stromal cell, Human umbilical cord mesenchymal stem cell, Stem cell spheroid