| Background:Intrauterine adhesion(IUA)can lead to abortion and infertility.The treatment of mesenchymal stem cells and their derived exosomes shows significant anti-fibrosis and endometrial regeneration potential in IUA treatment.However,the clinical application of traditional two-dimensional adherent culture mesenchymal stem cells(2D-MSCs)and their derived exosomes(2D-MSC-Exos)is limited due to the low cell activity and short residence time after transplantation,the low secretion of stem cell-derived exosomes and the unclear treatment mechanism.Combining mesenchymal stem cells with hydrogel for three-dimensional microencapsulated mesenchymal stem cells(3D-MSCs)culture can provide a new method to solve the above problems.Object:The microencapsulated culture system was constructed to explore the therapeutic effect of 3D-MSCs and their derived exosomes(3D-MSC-Exos)on IUA and further explore the possible mechanism.Methods:1.In the first part of the study,the microencapsulated stem cell encapsulation system was constructed and the stem cells were encapsulated and cultured.The microcapsules were characterized by scanning electron microscopy(SEM).Flow cytometry and CCK-8kit were used to detect the changes in the activity and proliferation of 3D-MSCs,and the changes in the multipotent transcription factors of 3D-MSCs were detected by flow cytometry,immunofluorescence and qRT-PCR.The model of damaged endometrial stromal cells(EndoSCs)was constructed to verify the therapeutic effect of 3D-MSCs and2D-MSCs on damaged EndoSCs.Then,the animal model of IUA was established and the effect of 2D-MSCs and 3D-MSCs on the endometrial structure of model animals was detected by HE staining.The endometrial fibrosis of model animals were detected by Masson staining and immunohistochemistry.Finally,the fertility recovery of model animals was testing by mating experiment.2.In the second part of the study,the exosomes derived from mesenchymal stem cells were first extracted and identified,then the damaged EndoSCs were constructed,and the therapeutic effects of 3D-MSC-Exos and 2D-MSC-Exos on the damaged EndoSCs were verified.Next,the animal model of IUA was established and the effect of 2D-MSC-Exos and 3D-MSC-Exoss on the endometrial structure of model animals was detected by HE staining.The CD31 expression of model animals were detected by immunohistochemistry.Finally,the fertility recovery of model animals was testing by mating experiment.3.Fibrotic EndoSCs was induced by TGF-β1,and the effect of 2D-MSC-Exos and3D-MSC-Exos on the fibrotic EndoSCs was verified.By analyzing the difference of miRNA expression in 3D-MSC-Exos and 2D-MSC-Exos and combining with relevant literature,miR-219-5p was selected as the research target,and the change of miR-219-5p expression was detected in clinical samples and fibrotic EndoSCs.The effect of miR-219-5p on fibrotic EndoSCs was verified by overexpression of miR-219-5p.Results:1.The SEM detection of microcapsules in the first part of the study showed that the microcapsules were regular in shape,with interconnected pores distributed on the surface,which could allow the entry and exit of therapeutic substances secreted by stem cells.The results of live cell/dead cell/apoptotic cell kit and flow cytometry showed that the viability of 3D-MSCs was good.The results of CCK-8 experiment showed that the proliferation ability of 3D-MSCs was significantly higher than that of 2D-MSCs.The results of flow cytometry,immunofluorescence and qRT-PCR showed that the expression of pluripotent transcription factors in 3D-MSCs was higher than that in 2D-MSCs.The experimental results of damaged EndoSCs showed that 3D-MSCs had a better effect on the morphology recovery of EndoSCs than 2D-MSCs.The flow cytometry showed that3D-MSCs could significantly reduce the apoptosis of damaged EndoSCs and increase their survival rate compared with 2D-MSCs.The mouse model of IUA was treated by intrauterine perfusion of stem cells.HE results showed that 3D-MSCs could better increase the thickness of endometrium and the number of glands than 2D-MSCs.Masson staining results showed that 3D-MSCs could better reduce the proportion of endometrial fibrosis than 2D-MSCs.Immunohistochemical results showed that 3D-MSCs could better reduce the markers of endometrial fibrosis than 2D-MSCs,and the results of the mating test suggest that 3D-MSCs have a better effect on the fertility restore of mice than2D-MSCs.2.In the second part of the study,the stem cell-derived exosomes were quantified and characterized.The results showed that the secretion of 3D-MSC-Exos was significantly higher than that of 2D-MSC-Exos.The experimental results of damaged EndoSCs showed that 3D-MSC-Exos had a better repair effect on the morphology of EndoSCs than2D-MSC-Exos.The flow cytometry showed that 3D-MSC-Exos could significantly reduce the apoptosis of damaged EndoSCs and increase their survival rate compared with2D-MSC-Exos.The results of western blot test suggested that 3D-MSC-Exos might have a therapeutic effect on endometrial damage through PTEN/AKT pathway.The mouse model of IUA was treated by injecting exosomes through the tail vein.The HE results showed that 3D-MSC-Exos could better increase the thickness of endometrium and the number of glands than 2D-MSC-Exos.The immunohistochemical results showed that 3D-MSC-Exos could better enhance the expression of endometrial vascular marker CD31 than 2D-MSC-Exos.The results of the mating test suggested that 3D-MSC-Exos could better restore the fertility of mice than 2D-MSC-Exos.3.The fibrotic EndoSCs was constructed by TGF-β1,and the results suggested that TGF-β1 treatment can reduce the migration ability of EndoSCs and significantly increase the expression of α-SMA and type I collagen.Exosomes treatment can increase the migration ability of fibrotic EndoSCs and reduce the expression of α-SMA and type I collagen,and the effect of 3D-MSC-Exos is better.Through the analysis of the difference of miRNA expression in 3D-MSC-Exos and 2D-MSC-Exos,it was found that the content of miR-219-5p in 3D-MSC-Exos was significantly higher than that in 2D-MSC-Exos.Through the analysis of the expression of miR-219-5p in the endometrium of clinical IUA patients and normal controls,it was found that the level of miR-219-5p in the endometrium of IUA patients was significantly lower than that of the normal control group.The level of miR-219-5p in fibrotic EndoSCs model and normal EndoSCs was also detected.The results showed that the expression of miR-219-5p in fibrotic EndoSCs was significantly reduced.Transfected with miR-219-5p mimics,the qRT-PCR results showed that the expression of miR-219-5p in fibrotic EndoSCs after transfection was significantly increased,and the overexpression of miR-219-5p could increase the migration ability of fibrotic EndoSCs and reduce the expression of α-SMA and type I collagen.Conclusions:1.The microenvironment formed by microcapsules is conducive to the growth and proliferation of stem cells,promotes the expression of pluripotent transcription factors,and increases the therapeutic effect on IUA by promoting the retention of stem cells at the damaged site.3D-MSCs can better restore the morphology of the endometrium,reduce its fibrosis,and promote the recovery of fertility,which can provide a new research direction for the treatment of stem cells in IUA.2.The microenvironment formed by microcapsules is conducive to the secretion of stem cells.3D-MSC-Exos is better than 2D-MSC-Exo in the treatment of IUA.It is a good choice for applying stem cell derived exosomes to treat IUA.3.The microenvironment formed by microcapsules will affect the type and content of substances secreted from stem cells.The expression of miR-219-5p in 3D-MSC-Exos is significantly increased.The therapeutic effect of 3D-MSC-Exos on endometrial fibrosis may function through miR-219-5p. |
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