Study On Treatment Of Intrauterine Adhesions In A Rat Model With Exosomes Derived From Menstrual Blood-derived Stromal Cells

Objective: Intrauterine adhesions are easily caused by induced abortion,spontaneous abortion and curettage,postpartum infections,and intrauterine surgery.It is the second leading cause of secondary infertility in Chinese women.Mesenchymal stem cell transplantation is widely used in damage repair and disease treatment.Among them,menstrual blood-derived stromal cells(MenSCs)with common characteristics of mesenchymal stem cells,are easy to obtain,rich in source and low in immunogenicity.MenSCs have broad development prospects in the field of stem cell regenerative medicine.Results in our previous studies confirmed the effectiveness of MenSCs in the treatment of intrauterine adhesions in severe IUA patients and in rat models.We found that MenSCs may exert some of the therapeutic effects through their paracrine mechanisms.In order to further explore the paracrine mechanism of MenSCs,we transplanted MenSCs-derived exosomes(MenSCs-Exos)into the uterus of IUA rat and investigated the therapeutic effects of MenSCs-Exos,and explored possible mechanisms for their effects.Methods: Exosomes in MenSCs serum-free culture supernatant were separated and extracted by ultracentrifugation.They were identified by transmission electron microscopy,nanoparticle tracking analysis and western blot technology.The IUA rat model was established by mechanical injury method.Rats were randomly divided into three groups and transplanted with PBS,MenSCs,and MenSCs-Exos respectively for treatment.Endometrial morphology,fibrotic deposition,and improvement of fertility of IUA rats were evaluated by pathological section staining,collagen expression detection,and pregnancy test after treatment.Immunohistochemical staining was used to detect the cell proliferation of endometrium after treatment.Three-dimensional imaging technology was used to observe the endometrial glands and vascular regeneration.Western blot was used to detect the expression of endometrial receptive protein after treatment.Results: MenSCs-derived exosomes were successfully isolated.MenSCs-Exos showed a typical round and cup-like structure with a complete membrane.NTA results showed that the particle size ranged from 30-150 nm.Western blot identified the expression of CD63 and CD81.MenSCs and MenSCs-Exos significantly promoted endometrial regeneration,increased the number of glands,and reduced endometrial collagen fiber deposition after the treatment.The results of pregnancy experiments showed that the number of embryos in the MenSCs group and MenSCs-Exos group was significantly higher than the control group.The results of immunohistochemistry showed that the MenSCs group and the MenSCs-Exos group got more epithelial cells with Ki67 positive expression when compared with the control group and the positive expression of CK-18 in cytoplasm also increased significantly.The results of three-dimensional imaging showed that MenSCsExos migrated around the regenerating glands and MenSCs migrated to the vicinity of blood vessels.Both of them promoted endometrial glands and blood vessel regeneration.The results of western blot showed that the endometrial receptive protein significantly increased after MenSCs and MenSCs-Exos treatment.Conclusion: MenSCs-derived with injury tropism,could effectively repair damaged endometrium,increase regeneration of endometrial glands and blood vessels,improve endometrial receptivity,and restore fertility in IUA rats.Our results demonstrated the important role of MenSCs-derived exosomes in the treating mechanism of MenSCs,and provided a theoretical basis for MenSCs-Exos as a cell-free therapy for endometrial regeneration in the future.