In Vitro Experimental Study Of Treatment Effect Of Human Amniotic Mesenchymal Stem Cells Combined With Estrogen On Intrauterine Adhesions

Objective: To study the effects of different concentrations of estradiol(E2)on the proliferation and apoptosis of human amniotic mesenchymal stem cells(h AMSCs)in vitro.Explore the conditions of h AMSCs differentiating into endometrial-like cells in vitro.Method:(1)HAMSCs were extracted by explant-attached method and enzyme digestion method.By flow cytometry and immunofluorescence were used to identify the h AMSCs at passage 3.HESt Cs were extracted by enzyme digestion.By immunofluorescence was used to identify the h ESt Cs.(2)Using different concentrations of E2 in h AMSCs.Cell counting kit-8(CCK8)method was used to measure the situation of h AMSCs proliferation of each group.Quantitative real-time polymerase chainreaction(q RT-PCR)was applied to measure the expression of the m RNA associated with pluripotency and the expression of apoptosis-related genes.The expression of apoptosis-related protein was measured by Western blotting(WB).(3)The condition of the induction medium and Transwell co-culture method were cultured for the h AMSCs at passage 3.After 7 days of culture,the expression of epithelial specific gene(CK7,EMA),mesenchymal specific gene(Vimentin)and decidualization marker gene(IGFBP-1)were detected by q RT-PCR.The expression of epithelial specific protein(CK18),mesenchymal specific protein(Vimentin)and decidualization marker protein(PRL)were detected by western blot.Results:(1)A huge amount of purified h AMSCs and h ESCs which could firmly proliferate were obtained through enzyme digestion method and passage for further research.(2)There was no significant difference between the the proliferation rate of the E2 group of each concentration in the 1-4 days and the control group,while the proliferation rate of each concentration E2 group was significantly higher than that of the control group after 4 days(P<0.05),and there was no significant difference among the concentration groups.The m RNA expression of stem cell related genes(Oct-4)and anti-apoptotic genes(Bcl-2)were significant higher in 1×10-6mol/l E2 group than in control group and other E2 concentration group(P < 0.05).And the expression of pro-apoptotic genes(Bax)m RNA was significant lower in 1×10-5mol/l E2 group than other E2 concentration group(P < 0.001).(3)The h AMSCs showed morphological changes from a spindle-shaped appearance to large fusiform cell in cytokines and cytokines +E2 group.The m RNA expression of CK7 and EMA were significant higher in cytokines group than in control group(P<0.05).The protein expression of CK18?PRL were significant higher in cytokines and E2+ cytokine group than control group(P<0.01).Nevertheless,the protein expression of Vimentin was significant lower in cytokines and E2+ cytokine group than in control group(P<0.01).The expression of CK7 and IGFBP1 m RNA were up-regulate(P<0.05),while the expression of Vimentin m RNA was down-regulated in co-culture group than control group(P<0.05).Conclusion:(1)A huge amount of h AMSCs could be obtained by the modified enzyme digestion method.(2)E2 can promote the proliferation of h AMSCs,proliferative effect was time dependency.And high concentration of E2 group(10-5mol/l)could promote apoptosis,however higher concentration of E2 group(10-6mol/l)could maintain activity of h AMSCs and protect pluripotency.(3)HAMSCs could differentiate into endometrium-like cells in the microenvironment provided by cytokines(TGF-?1,EGF and PDGF-BB)or co-cultured with h ESt Cs.However,the effect of E2 on this differentiation is not significant.