| Background: Intrauterine adhesion(IUA),also known as Asherman syndrome,is a common reproductive organ dysfunction disease that seriously threatens women’s physical and mental health.The destruction of the basal layer of the endometrium caused by the operation of the uterine cavity is the main reason for the formation of IUA.Due to the increase in intrauterine surgery,the incidence of IUA in my country has been increasing year by year,and it has now become the second leading cause of secondary infertility.Hysteroscopy to remove intrauterine adhesions is the main IUA treatment method.However,for some patients with moderate to severe IUA,adhesion recurrence and low pregnancy rate are problems that need to be solved urgently in clinical treatment.With the development of regenerative medicine,it has been confirmed that mesenchymal stem cells play an important role in tissue damage repair.The previous research of the research group confirmed that the transplantation of autologous menstrual blood-derived stromal cells(MenSCs)significantly promoted endometrial regeneration and uterine cavity morphology recovery in patients with severe IUA,and some patients succeeded in pregnancy after endometrial repair.However,the timeliness of intrauterine infusion of MenSCs needs to be improved.How to improve the colonization and treatment efficiency of MenSCs is a key issue that needs to be solved in the clinical transformation of stem cell therapy for IUA.Platelet-rich plasma(PRP)is an artificially prepared peripheral venous blood-derived product,rich in a variety of growth factors,and has been proven to promote cell functions,interact with the original extracellular matrix of tissues,and regulate stem cell microbes.The environment is conducive to cell proliferation and wound healing.Mesenchymal stem cells from a variety of tissues all show function promotion phenomena such as proliferation,differentiation and recruitment after PRP treatment.In addition,PRP exhibits a liquid-gel biphasic dynamic fibrin scaffold structure after activation,which is sufficient to maintain its biological activity in the body for a long time and improve the biological efficacy of transplanted cells in stem cell therapy.It is an ideal stem cell scaffold.material.Therefore,this study intends to clarify the promotion effect of PRP on MenSCs,explore the key effect factors in PRP,and use the rat IUA disease model to explore the therapeutic effect of MenSCs combined with PRP transplantation on IUA on the basis of previous studies,and to further explore the repair of endometrial injury by MenSCs The molecular mechanism of the disease is studied at the tissue level,cell level,and molecular level to evaluate new strategies for stem cell treatment of endometrial injury.Methods and Materials:(1)In vitro cell experiments were used to explore the promotion effect of PRP on the biological function of MenSCs: CCK8,immunofluorescence,flow cytometry,RT-PCR and Western-blot were applied to clarify the regulatory effect of PRP on MenSCs;(2)Animal experiments were used to explore the therapeutic effect of MenSCs combined with PRP transplantation on IUA.(2)To investigate the therapeutic effect and mechanism of MenSCs combined with PRP transplantation on IUA by animal experiments: Immunohistochemistry,immunofluorescence,RT-PCR and Western-blot were applied to detect the changes of endometrial thickness,fibrosis and other pathological indicators and the expression of endometrial stem cell markers in IUA uteri after treatment at the tissue and molecular levels;pregnancy experiments were used to investigate the improvement of pregnancy outcome of MenSCs combined with PRP transplantation in IUA rats.(3)In vitro experiments with clinical samples,using CCK8,immunofluorescence,flow cytometry and Western-blot techniques,clarified that PDGFBB was the key effector of PRP on MenSCs.The molecular pathway of the action of MenSCs on improving the endometrium of IUA was verified in combination with the results of the second part,and the reversal of endometrial fibrosis by MenSCs was further clarified.Results: The first part of the results shows that activated PRP has a positive regulatory effect on the biological functions of MenSCs.PRP can significantly promote the proliferation of MenSCs,increase the proportion of S-phase cells,inhibit cell apoptosis related to in vitro culture,increase the expression of MenSCs markers SUSD2,CD146 and CD105,and promote the osteogenic differentiation and adipogenic differentiation of MenSCs.The second part of the results shows that PRP has a synergistic effect on MenSCs treatment of rat IUA model.PRP combined transplantation significantly improved the colonization efficiency of MenSCs in the endometrium of IUA rats,and increased the expression of endometrial stem cell marker PDGFR.The therapeutic effect of MenSCs combined with PRP is significantly better than that of MenSCs transplantation alone or PRP injection.MenSCs combined with PRP transplantation can significantly promote endometrial proliferation,promote endometrial stroma,glands and angiogenesis,and increase the expression of CK18 and CD31 in the endometrium of IUA rats Level,improve endometrial fibrosis and reduce collagen deposition,significantly increase the pregnancy rate and live birth rate of IUA rats;paracrine effect may be an important way for MenSCs to treat endometrial injury,and PRP can improve the intrauterine human in rats.Mesenchymal stem cells secrete factors IGF-1,SDF-1 and TSP-1 gene expression levels;MenSCs treatment significantly regulates the expression of key molecules CTGF,WNT5 A and GDF5 downstream of the Hippo pathway,and PRP has a promoting effect on the regulation of MenSCs pathway.The third part of the results shows that,compared with normal MenSCs,PDGFR in MenSCs of IUA patients is low,and exogenous PDGFBB has a significant positive regulatory effect on MenSCs of IUA patients,which is close to the results of PRP culture in the first part.PDGFBB promotes MenSCs Proliferate and migrate,increase the proportion of S-phase cells,activate the Akt/NFκB pathway and increase the expression of P65 and nuclear translocation.The conditioned medium of MenSCs significantly reduced the expression of YAP and CTGF in the endometrium of IUA patients,and PDGFBB pretreatment can promote this phenomenon.Conclusion: This study confirmed that PRP has a promoting effect on the biological function of MenSCs,established a combined MenSCs-PRP transplantation system,clarified that PRP can be used as a MenSCs transplantation adjuvant to improve the therapeutic efficiency of MenSCs for IUA,found that Hippo pathway may be an important molecular pathway for MenSCs to reverse endometrial fibrosis It was further confirmed that PDGFBB is a key effector of PRP acting on MenSCs to improve the biological function of MenSCs by activating AKT/NFκB pathway,and paracrine secretion may be an important mode of action for MenSCs to play a role in the treatment of endometrial injury.This study initially elucidates the key molecular mechanism of endometrial injury repair and improvement of IUA by transmural stem cells,which provides an experimental basis for the clinical application of MenSCs combined with PRP in the treatment of IUA,and lays a solid theoretical foundation for the further clinical promotion of MenSCs in the treatment of IUA. |
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