| Objective: To explore and construct the intrauterine adhesion(IUA)animal model in pregnant mice,to provide a more ideal animal model for the study of the treatment of intrauterine adhesion.To investigate the repair effect of human amniotic mesenchymal stem cells(h AMSCs)transplantation on intrauterine adhesions in pregnant rats.Methods: 1.Isolation and extraction of human amniotic mesenchymal stem cells from human amniotic membrane by enzymatic digestion,their phenotypic characteristics were identified by cell immunofluorescence and flow cytometry,and their proliferation activity was detected by CCK-8 method.2.Randomly divided the Twenty-four female SD rats into four groups: A,B,C and D,with 6 rats in each group.Group A was bred routinely;Groups B,C,and D were reared with male mice and caged to make them pregnant,and the IUA model was established by mechanical curettage after abortion on the 5.5th to 6.5th day of pregnancy.Group A: open abdomen,exposed uterus,no curettage.Group B: curettage after abortion,1 estrous cycle after curettage.Group C: curettage after abortion,2 estrous cycles after curettage.Group D:curettage after abortion,3 estrous cycles after curettage.At the end of the observation,the rats in each group were sacrificed,and bilateral uterine specimens were collected,which were modeled and judged by HE staining and Masson staining.3.36 SD female rats were randomly divided into control group,model group and transplantation group,with 12 rats in each group.The model group and transplantation group underwent mechanical curettage after abortion to establish an IUA model,and the transplantation group underwent h AMSCs transplantation 2 estrous cycles after modeling.Two weeks after transplantation,6 rats in each group were randomly sacrificed,and the repair effect of h AMSCs transplantation on intrauterine adhesions in pregnant mice was evaluated by HE staining,Masson staining,and immunohistochemistry.The remaining rats in each group were randomly combined with male rats at a ratio of 3:1.On the 14.5th day of pregnancy,the bilateral uterus was taken out to count the number of embryos implanted,and the effect of h AMSCs transplantation on pregnancy ability was analyzed.Results: 1.After about 24 hours of culture,the primary h AMSCs adhered to the wall and grew in a round or polygonal shape.The shape of the passaged h AMSCs was relatively consistent,growing in a long fusiform swirling shape,and arranged closely.Cell immunofluorescence showed that the third generation of h AMSCs expressed vimentin but not CK19.Flow cytometry showed that P3 h AMSCs had high expression of CD105(94.39%),CD90(99.98%)and CD44(99.92%);Low expression of CD19(0.11%)and CD45(0.17%).The CCK-8 assay showed that the growth characteristics of h AMSCs were consistent with that of most mesenchymal stem cells.2.Compared with group A,the number of glands in group B,C,and D decreased,and the degree of fibrosis was aggravated,with difference was statistically significant(P<0.001).Compared with group B,the number of glands in groups C and D decreased and the degree of fibrosis was aggravated,with difference was statistically significant(P< 0.001).Compared with group C,the number of glands and the degree of fibrosis in group D had no statistical significance(P>0.05).3.Compared with the model group,the uterine cavity morphology of the transplantation group was significantly restored,the number of endometrial glands increased and the morphology of the endometrial cavity was restored,the degree of fibrosis was reduced,the expression of TGF-β1 decreased,the expression of MMP-9 and VEGF increased,and the number of embryo implantation was increased.Conclusion: 1.Mechanical curettage after abortion can build a stable and effective animal model of intrauterine adhesions in pregnant rats.2.h AMSCs transplantation can repair the intrauterine adhesion endometrial structure of pregnant mice,increase the number of glands,reduce fibrosis,attenuate the degree of increased expression of TGF-β1,alleviate the degree of decreased expression of MMP-9 and VEGF,restore the shape of the uterine cavity and improve the quality of the uterus.Improve pregnancy ability. |
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