Isolation And Validation Of Human Endometrial Mesenchymal Stem Cells And The Research About The Effect Of Growth Factor On Differentiation Into Ligament Fibroblasts

Objective:1.Isolation of hEMSCs in vitro,and discuss whether hEMSCs possesses the characteristic of mesenchymal stem cells.2.To investigate the effect of basic fibroblast growth factor（bFGF）and transform growth factor-β₁（TGF-β₁）on the differentiation of hEMSCs into ligament fibroblasts.Methods:1.Isolation and identification of hEMSCs in vitro:separation and purification of hEMSCs by enzyme digestion combined with repeated adherence method.Cell morphology was observed under inverted contrast microscope.Flow cytometry was used to detect the positive rate of cell surface specific antigen.The differentiation of hEMSCs into adipocytes,osteoblasts and chondroblasts by specific induction medium in vitro.The effects of hEMSCs into adipocytes,osteoblasts and chondroblasts differentiation were determined by oil red O staining,alizarin red staining and Alcian blue staining.MTS assay were performed to evaluate the rate of cell proliferation and charts the growth curve of the cells.2.Using the fourth generation of hEMSCs,bFGF,TGF-β₁,and bFGF+TGF-β₁ were added to the medium as the experimental group,and the normal medium were added as the control group.After 3 days of culture,the morphology of each group was observed under inverted contrast microscope.The proliferation rate of each group was detected by 12、24、36、48、60 h using MTS assay.After 3 days of culture,real-time quantitative PCR was used to detect the relative mRNA expression levels of collagen type I.The protein expression of collagen type I were detected by immunefluorescence staining.3.Data analysis were performed using GraphPad Prism 7.Data was presented in figure and text as mean±standard deviation（?±）.Differences between the two groups were compared by t test,withα=0.05 as the test level,and P<0.05 indicates that the difference was statistically significant.Results:1.Under the inverted microscope,hEMSCs were observed to have uniform cell morphology after multiple passages,which were fibroblast-like and vortex-like or parallel-arranged.The surface specific antigen molecules of the fourth generation hEMSCs were positive for CD73,CD90 and CD105 by flow cytometry,which were 99.9%,97.3%and99.7%,respectively.The surface markers of hematopoietic stem cells were negative for CD34,CD45,CD11b,CD19 and HLA-DR.,which was 1.7%.Oil red O staining showed that lipid droplets were observed in the cytoplasm after adipogenic induction;alizarin red staining showed multiple mineralized nodules outside the cells after osteogenic induction;alimin blue staining showed:a large amount of glycosaminoglycan is secreted into the cell matrix after chondrogenic induction.The cell growth curve shows that the cell proliferation ability is high.The above results confirmed that the obtained cells conformed to the characteristics of mesenchymal stem cells.2.After intervention with bFGF,TGF-β₁ and bFGF+TGF-β₁:（1）After 3 days of culture,the morphology of the cells was observed under inverted contrast microscope.The results showed that compared with the control group,the bFGF group and the bFGF+TGF-β₁ group were more elongated and showed a"dendritic"shape,which tends to be fibroblasts.The morphology of cells in the TGF-β₁ group was broadened and showed a tendency to spread.（2）The cell proliferation rate of each group of 12、24、36、48、60 h of cultured valuated by MTS assy showed that the proliferation rate of bFGF group,TGF-β₁ group and bFGF+TGF-β₁ group was significantly faster than that of the control group,the difference was statistically significant（P<0.05）;bFGF+TGF-β₁ group proliferation rate was more obvious than bFGF group,TGF-β₁ group,the difference was statistically significant（P<0.05）;suggesting that bFGF and TGF-β₁ can be promote cell proliferation,and the combination of the two agonists promotes the cell proliferation more significantly.（3）Real-time quantitative PCR results after 3 days of culture showed that compared with the control group,the expression of collagen type I mRNA was down-regulated in bFGF group,and the expression of collagen type I mRNA in TGF-β₁ group and bFGF+TGF-beta1 were up-regulated,the difference were statistically significant.（P<0.05）,but the up-regulated trend of collagen type I mRNA in bFGF+TGF-β₁ group expression up-regulated trend was small,the differences wsa statistically significant（P<0.05）.（4）Immunofluorescence staining results showed that collagen type I was mainly distributed in the cytoplasm.Compared with the control group,the expression of collagen type I in bFGF group was significantly decreased,and the expression of collagen type I in TGF-β₁ group and bFGF+TGF-β₁ group were increased,but the increased degree of collagen type I in bFGF+TGF-β1 group was small.Conclusion:1.hEMSCs have MSC characteristics and can be used as a source of ligament tissue engineering seed cells.2.bFGF can promote hEMSCs cell morphology to be fibroblasts-like cell.The combined effects of bFGF and TGF-β₁ to promote the proliferation of hEMSCs were more obvious.bFGF and TGF-β₁ have a certain antagonistic effect on the synthesis of collagen type I.bFGF has the effect of inhibiting the synthesis of collagen type I,while TGF-β₁ has the effect of promoting the synthesis of collagen type I.The combination of bFGF and TGF-β₁ makes the synthesis and degradation of collagen type I in a state of dynamic equilibrium,and generally increases the expression of collagen type I,which promotes the differentiation of hEMSCs to ligament fibroblasts.