Bone Marrow Mesenchymal Stem Cell-derived Exosomes Promote Proliferation And Collagen Expression Of Fibroblasts Of The Main Ligament In Patients With Pelvic Organ Prolapse

Pelvic organ prolapse(POP)is a type of gynecological disease caused by pelvic organ decline and displacement caused by weak pelvic floor support structure.The abnormal structure and function of the pelvic floor support system are closely related to its pathophysiology.Numerous studies have shown that exosomes derived from mesenchymal stem cells have a repairing effect on a variety of tissues,including connective tissue.ObjectiveBy studying the effect of the homogenous BMSC-Exo on primary fibroblasts of the uterine main ligament in patients with severe POP,we further explored the promoting effect of BMSC-Exo on the proliferation of these cells and the expression of collagen and its potential mechanism of action,and provided possible experimental basis and theoretical basis for non-surgical biotherapy of POP in clinical practice.Materials and methods1 Bone marrow lavage fluid was extracted from sacroiliac tissues,which were taken from patients with accidental fractures.Using the cell adherence method,cells from bone marrow lavage fluid were purified,and the morphology of their subculture was observed using an inverted microscope.Their specific surface marker proteins were identified by Flow Cytometry.2 Bone marrow mesenchymal stem cells-secreted exosomes(BMSC-Exo)were isolated and purified from the culture medium of the 3rd generation BMSCs by differential ultracentrifugation.The morphology of BMSC-Exo was observed using a transmission electron microscope.Nanoparticles analyze(NTA)was performed to analyzed particle size distribution and particle concentration.Western blot was performed to identify exosomal specific marker protein purified BMSC-Exo.3 Uterine main ligament tissues were taken from patients with postmenopausal POP according to POP-Q scores for isolation of hCLF.The hCLF was isolated and adhered as the research object,and its morphology and distribution were observed by an inverted microscope.According to the cell MTT test the cell proliferation activity of BMSC-Exo with different concentration gradients on primary hCLF for different time periods was measured to select the optimal concentration and time.4 After using BMSC-Exo with this concentration on primary hCLF for a certain period of time,qRT-PCR and Westernblot methods were used to evaluate the synthesis of hCLF collagen,its metabolic enzymes,and important molecules in related pathways from transcription and protein expression levels,respectively.Result1 After subculture of the isolated BMSCs,the BMSCs are in the shape of long spindle or polygonal under a light microscope,and they were arranged in a vortex.Flow cytometry showed that both CD44 and CD90 were highly expressed,with positive rates of 99.74%and 97.16%respectively,while CD34 and CD45 were almost negatively expressed.2 Electron microscope analysis showed that BMSC-Exo had centrally depressed coaster shape and a membrane structure.The mean diameter of BMSC-Exo was 78.12±34.45 nm,and the particle size distribution was concentrated at 50?110 nm.Western blot analysis confirmed that HSP70,CD63,and CD9 were highly expressed in BMSC-Exo,while calnexin was hardly expressed.3 The distribution of primary hCLF in POP patients was sparse or polymorphous,mainly in the shape of spindle or spindle,and arranged in an irregular weave.After MTT test,it was found that the proliferation activity of primary hCLF was increased after 24,48 and 72 hours with 20 ?L of BMSC-Exo at 10,20,30 and 40 ng/mL,showing A certain degree of time-dependent property,The statistical results showed that 30 ng/mL effect was the most significant difference between groups for 48 hours.4 After 48 hours of 30 ng/mL of BMSC-Exo treatment on primary hCLF,qRT-PCR results showed that the mRNA expression of COL1A1,COL3A1,MMP-9 and TIMP-1 was significantly increased.Moreover,compared with the control group,TGF-?1 and IL-6,which are reported to involve in pathway of collagen fibrogenesis,were significantly increased in BMSC-Exo treatment group.Western blot results showed that except for the reduction of MMP-9 protein,the remaining 5 proteins were higher expressed in BMSC-Exo treatment group than that in control group.The ratio of type?/? collagen was increased in BMSC-Exo treatment group.ConclusionAllogeneic BMSC-Exo can promote the synthesis of type ? and type ? collagen by primary hCLF in the pelvic ligament of POP patients,and reduce the enzymatic activity of collagen by down-regulating MMP-9 and increasing the expression of TIMP-1.Further,remodeling of pelvic floor connective tissue ECM through the above action is conducive to improving the strength,toughness and compliance of ligaments.One possible mechanism is that the process is realized by BMSC-Exo through activating the TGF-?/Smad signaling pathway and the two-way promotion effect with the cytokine IL-6,and regulating the local immune microenvironment.