Effects Of 17-β Estradiol On Olfactory Epithelial Stem Cells Proliferation And Differentiation

Objective:To investigate the effect of estrogen on the proliferation and neural differentiation of olfactory epithelial stem cells,improve the existing culture methods of olfactory epithelial stem cells,and to investigate the expression of related signaling pathways,so as to provide potential explanations for the treatment of olfactory disorders by estrogen..Methods:1.Primary olfactory epithelial stem cells culture of female rat was performed and primary cells were identified by immunofluorescence.2.Olfactory epithelial stem cells were treated with 17-β estradiol with concentrations of 10-5M,10-6M,10-7M,10-8M,10-9M,10-10M,respectively.Cell proliferation was detected by CCK-8 assay,and the concentration that significantly promoted the proliferation of olfactory epithelial stem cells was selected for subsequent experiments.3.Cells were divided into estrogen group and control group,and the nerve differentiation was identified by immunofluorescence after induction of differentiation for 7 days.4.Cells were divided into estrogen treatment group,estrogen+ICI 182780 group,estrogen+DKK1 group and control group.The cell proliferation and olfactory-sphere formation were detected by CCK-8 assay and neurosphere assay,respectively.5.Statistical analysis was performed using GraphPad Prism 9 and SPSS19.0 software.One-way analyses of variance were used to compare the means of the groups and t-test was used for pairwise comparison between groups.Result:1.Characteristics of primary culture of olfactory epithelial stem cells:at culture day 6,part of olfactory epithelial cells adherent to wall and cell colonies emerged,and the olfactory epithelium consisted of adherent cells with heterogeneous morphology.At culture day 9,the colonies increased continuously,and most of the cells grew like fibroblasts like mesenchymal stem cells,with spindle-shaped surface and a few slender protuberances.The cells were homogeneous and swirled after passage.In proliferation culture,the cells began to adhere to the wall and grew like colonies,some cells migrated to the center of the colony,and olfactory-spheres were observed at 7 days.Immunofluorescence showed that adherent cells and olfactory-spheres expressed Nestin,a marker of neural stem cells.2.CCK-8 assay showed that,compared with the control group,estrogen concentrations of 10-6M,10-7M and 10-8 M could promote the proliferation of olfactory epithelial stem cells,of which 10-7M had the most obvious effect(P<0.01).Estrogen+ICI 182780 group and estrogen+DKK1 group significantly inhibited this effect(P<0.01).3.Neurosphere analysis showed that,compared with the control group,the diameter of olfactory-spheres in estrogen group were larger(P<0.01),and the quantity was more.;The diameter of estrogen+ICI 182780 group and estrogen+DKK1 group(P<0.01)and the quantity was less.4.Immunofluorescence results showed that,compared with the control group,estrogentreated olfactory epithelial stem cells had a higher proportion of neurons post differentiation induction for 7 days(P<0.01).Conclusion:1.Estrogen could promote the proliferation of olfactory epithelial stem cells and the formation of olfactory proliferating spheres,and the optimal concentration is 107M.2.Estrogen can promote neural differentiation of olfactory epithelial stem cells.3.The mechanism of estrogen promoting proliferation of olfactory epithelial stem cells,the formation of olfactory-spheres and neural differentiation may be related to the ERWnt signaling pathway...