Study On Differentiation Menstrual Blood-derived Mesenchymal Stem Cells And The Effects On Human Endometrial Stromal Cells

Objective: The purpose of the study is to compare the separation and extraction of menstrual blood-derived mesenchymal stem cells by adherent method and density gradient centrifugation method;at the same time,to explore the action of Men SCs on endometrial stromal cells,providing basis for the clinical application.Methods:1.The menstrual blood of healthy women younger than 35 years old was collected by aseptic menstrual cup on the 2nd-4th day.The Men SCs were extracted by Ficoll density gradient centrifugation and adherent method,and identified by flow cytometry.2.Growth factor and 17-?-estradiol valerate were added to construct the microenvironment for the differentiation of Men SCs into endometrial cells.Vimentin and Cytokeratin were detected by immunofluorescence to evaluate whether the Men SCs could be transformed into ESCs and epithelial cells.3.Endometrial tissues were collected from patients undergoing hysteroscopic curettage,ESCs were isolated by secondary mesh screening and cultured,and identified by immunofluorescence.4.Through the indirect co-cultured system of Men SCs and ESCs,CCK-8 method was used to evaluate the proliferation of normal ESCs and the expression of Vimentin and VEGF was detected by Western blot,observing the effect of Men SCs on the expression of the two proteins.Results: 1.The positive rates of cell surface markers extracted by Ficoll density gradient centrifugation were 83.88%?98.69%?98.78%?99.80%,and the positive expression rates of surface markers were 61.39%?65.15%,99.66%?100.00%.The expression of HLA-DR,CD34 and CD45 on the surface of menscs extracted by two methods were all negative;2.After induction by growth factors and 17-?-estradiol valerate,the morphology of Men SCs began to change from short to long shuttles,and the morphology tended to long shuttles of ESCs,showing clump-like and vortex-like growth.Vimentin and CK were positive;3.The absorbance value of endometrial stromal cells cultured with Men SCs medium for 24 h,48 h and 72 h was significantly higher than that of endometrial stromal cells cultured alone.The expression of Vimentin and VEGF in the experimental group was higher than that in the control group,with significant difference(P<0.05).Conclusions:1.Higher purity of Men SCs can be obtained by Ficoll density gradient centrifugation and adherent method,and the purity of Men SCs obtained by Ficoll density gradient centrifugation is higher.2.Men SCs can differentiate into ESCs and epithelial cells under the induction of growth factors and 17-?-estradiol valerate.3.Men SCs can promote the proliferation of human ESCs,enhance the expression of VEGF,and improve endometrial receptivity.