Human Amniotic Mesenchymal Stem Cells Transplantation Combined With Estrogen In The Treatment Of Intrauterine Adhesions:A Rat Experimental Study

Objective:After establishing rat intrauterine adhesion(IUA)models by mechanical and infective double injury method,to explore the effect of amniotic mesenchymal stem cells(h AMSCs)with/without estrogen on IUA and its possible mechanism.Methods:h AMSCs were extracted by enzymatic digestion,in order to identify h AMSCs,the expression of CD29,CD90,CD34,HLA-DR was detected by flow cytometry and immunofluorescence was used to analyzed vimentin(VIM),keratin(CK19).The third generation of h AMSCs was stained with PKH26 and the instant labeling rate was calculated in vitro.The activity and proliferative capacity of h AMSCs were detected by CCK-8 assay,apoptosis and cell cycle were detected by flow cytometry to observe the effect of PKH26 stain on the biological characteristics of h AMSCs.2.Sixty female SD rats were randomly divided into five groups,which were control group(A),IUA group(B),estrogen group(C),hAMSCs transplantation group(D),h AMSCs and estrogen combination group(E).The rat IUA model was established by mechanical and infective double injury method,after one week,the uteruses of 6 rats in each group were subjected to HE staining and Masson staining to determine whether the models were successfully established.The remaining rats in groups C,D,and E were given appropriate treatment:each rat in group C was given0.1mg/kg oral estrogen for 5 days;the rat in group D was transplanted with1 mL PKH26-labeled h AMSCs(approximately 2x10~6)via tail vein;the rat in group E was given both of hAMSCs injection and oral estrogen.After 2weeks of treatment,the uterine tissues of each group were collected.HE staining was used to calculate the number of glands and Masson staining to analyse the fibrosis in the endometrium.The distribution of PKH26-labeled h AMSCs in the endometrium were observed with fluorescence confocal microscope.The changes of VEGF,CK in the uterine tissues were measured by immunohistochemistry.Results:1.The morphology of h AMSCs tended to be stable and was spindle-shaped after three-passage cultivation.The flow cytometry showed that h AMSCs highly expressed CD29 and CD90,and almost did not express CD34 and HLA-DR.The immunofluorescence test showed that h AMSCs mainly expressed mesenchymal cell marker VIM but not epithelial cell marker CK19.The immediate labeling rate of h AMSCs stained by PKH26 was approximately 100%,and the PKH26 marker had no significant effect on the biological characteristics such as activity,proliferation,apoptosis,and cycle of h AMSCs.2.The number of glands was decreased significantly and fibrosis was increased in the endometrium on IUA(P<0.05).Compared with group B,the number of glands in each treatment group(C,D,E group)increased to varying degrees,the degree of endometrial fibrosis decreased,the expression of VEGF and CK increased,among which group E was the most obvious,basically returning to the normal level of Group A.Scattered red fluorescence was observed in groups D and E,mainly in the rat endometrium by the fluorescence confocal microscope.Conclusion:The PKH26 labeling technique is a safe and effective method to track the distribution and migration of h AMSCs in vivo.A stable animal model of IUA can be established successfully using mechanical and infection double injury method.hAMSCs transplantation,when added to oral estrogen,is the best effective alternative on endometrial regeneration after injury in rats.It is speculated that h AMSCs secrete VEGF to induce blood vessel regeneration,and estrogen provides a good microenvironment for the induction of h AMSCs,so that the h AMSCs activity is maintained at a high level to promote the repair of endometrium.