The Role Of Endometriosis-derived Exosomes In The Pathogenesis Of Endometriosis

Background:Endometriosis(EMS)is a frequently-occurring disease in women of childbearing age.The main symptoms include infertility and various pelvic visceral pain.The recurrence rate after EMS treatment is high,which brings great physical and mental pain to patients.The existing pathogenesis theory includes retrograde menstruation,inmmunology,metaplasia and Mullerian remnants.Exosome is a 30-100 nm small vesicle that plays an important role in cell-cell communication.Early studies suggested that exosome only performs protein transport functions,and later discovered that exosome also transports nucleic acids including mRNA and miRNA,which are involved in intercellular communication.It has been shown to be involved in the progression of many diseases.Endometriosis patients and animal models have confirmed a series of pathological changes in the eutopic endometrium.Based on the theory of menstrual blood flow,we speculate that as a macrovesicles that can carry information,its diameter is very small,so that it can easily flow back into the abdominal cavity,which affects the microenvironment of the abdominal cavity and accelerates the progression of the disease.Part ? Identification and characterization of EMS-exosomesObjective:1.Construct a mouse endometriosis model to identify and compare exosomes secreted by endometrial stromal cells in model mice and normal mice;2.Verify the clinical collection of exosomes secreted by EMS and normal endometrial stromal cells in the endometrial samples of patients and identify and compare them.Materials and methods:1.Establish a model of mouse endometriosis by intraperitoneal injection of endometrial debris.Mouse EMS animal models were validated by HE staining(hematoxylin-eosin staining)and immunofluorescence staining.The endometrial stromal cells of endometriosis and normal mice were isolated and cultured in vitro,and the cell culture supernatant was collected;2.Intraoperative resection of endometriosis and normal endometrium,isolation and culture of endometrial stromal cells in vitro,verification of human-derived mesenchymal cells by immunofluorescence,culture of cells and collection of culture supernatant;3.Based on previous studies,improve the separation method of exosomes,separate exosomes by differential centrifugation,and identify the exosomes by electron microscopy,particle size analysis and Western blot.Including the size of the exosomes,morphological analysis.Results:1.Exosomes secreted by endometrial stromal cells derived from mouse endometriosis model are similar in morphology to exosomes secreted by normal mouse endometrial stromal cells;2.Endometrial stromal cells of endometriosis patients can secrete exosomes with a diameter of approximately 100 nm,which are not morphologically altered relative to exocrine secreted by normal endometrial stromal cells.Part ? EMS-exosomes promote neuroangiogenesisObjective:1.To investigate whether exosomes derived from endometrial stromal cells in EMS patients can be swallowed by DRG(spinal dorsal root ganglion)nerve cells and HUVEC(umbilical vein endothelial cells);2.To investigate whether exosomes derived from endometrial stromal cells in EMS patients can promote neuroangiogenesis;3.Whether the role of promoting angiogenesis is still present after GW4869 inhibits secretion of exosomes.Materials and methods:1.Exosome derived from endometriosis was labeled with PKH67 green fluorescently,and co-cultured with DRG nerve cells and umbilical vein endothelial cells.Neurons and endothelial cells were labeled by immunofluorescence to observe the phenomenon of exocytosis.The green-labeled exosomes were co-cultured with DRG nerve cells and endothelial cells,and the phagocytosis of exosomes was detected by flow cytometry at different time points;2.The effect of exosomes on axon growth of nerve cells was detected by immunofluorescence.The endogenous exogenous endosome was co-cultured with endothelial cells,and its effect on the formation of umbilical vein endothelial cells was observed under a microscope.3.After inhibiting the secretion of exosomes by GW4869,verify whether the role of neuroangiogenesis still exists.Results:1.Exosomes can be engulfed by DRG neurons and endothelial cells,and the rate of phagocytosis increases with the incubation time;2.Exosomes secreted by endometrial stromal cells of endometriosis can play a role in promoting neuroangiogenesis compared with exosomes derived from normal endometrial stromal cells,and inhibit exocrine secretion pathways.The role of promoting neuroangiogenesis disappears.Part ? EMS-exosomes remodel polarization of peritoneal macrophages and promote disease progression.Objective:1.In vitro experiments to verify the effects of exosomes derived from the endometriosis model in mice on the polarization and phagocytosis of RAW264.7(macrophages);2.In vivo experiments to verify the effects of exosomes derived from the endometrnosis model in mice on the polarization and phagocytosis of mouse peritoneal macrophages,and to explore the role of endocrine-derived exosomes in disease development.Materials and methods:1.In vitro experiment:we injected endometrial fragments to establish a model of endometriosis in mice.After successful modeling,endometrial stromal cells were isolated,exosomes were isolated and macrophage cultured,and immunofluorescence was performed.Detection,polarization analysis of macrophages by RT-PCR and flow cytometry;2.In vivo experiments,mice were injected intraperitoneally with endometriosis-derived exosomes for 7 days.The phagocytosis and polarization status of peritoneal macrophages were detected by flow cytometry.After successful modeling,macrophages were detected by immunofluorescence.The infiltration state of the cells and the size,number,and weight of the ectopic lesions.Results:1.Mouse endometriosis model Endometrial stromal cell-derived exosomes can induce macrophage polarization to M2,attenuating macrophage phagocytosis;2.After intraperitoneal injection of exosomes,the peritoneal macrophages of the mice changed correspondingly,the phagocytic ability decreased,and the polarization state changed.This change in peritoneal macrophages simultaneously increases the weight and volume of ectopic lesions.