| BackgroundPremature ovarian insufficiency(POI)is a clinical syndrome characterized by oligomenorrhea or amenorrhea for at least 4 months,elevated follicle-stimulating hormone levels(FSH,> 25 m IU/ml on two occasions > 4 weeks apart)and low estradiol(E2)levels in women before the age of 40 years,seriousiy,performance for decreased fertility even infertility.Currently,hormone replacement therapy,melatonin,DHEA,diet and exercise,which are commonly used in clinic,cannot fundamentally improve the ovarian function and enhance the fertility of patients.Therefore,finding new treatment methods to improve the ovarian function of patients and improve their fertility is an important problem to be solved urgently in clinical practice.Mesenchymal Stem Cells(MSCs)are adult Stem Cells,with multi-directional differentiation potential and self-renewal ability,and the world has a lot of animal experiments and clinical trials prove that MSCs can repair the damaged ovarian function by promoting follicular,restore hormone levels.However,the tumorigenicity and immunogenicity that MSCs transplantation causes have become a barrier to its clinical application.Exosomes derived from Mesenchymal Stem Cell(MSC-EXO)is a kind of nanoscale extracellular vesicles MSCs secrete.According to domestic and foreign research reports,MSC-EXOs has therapeutic effect on many diseases,such as reducing myocardial ischemia-reperfusion injury,promoting neurological recovery after stroke and wound healing,and can play the role of the similar to stem cells.MSC-EXOs,as a kind of cell free treatment strategy,can avoid many problems include immunogenicity and oncogenicity caused by stem cells transplantation,which brings great advantages for the wide application of MSC-EXOs.At present,there are few reports on the experimental study on POI by exosomes derived from mesenchymal stem cells at home and abroad,and whether MSC-EXOs has therapeutic effect on POI ? Therefore,the purpose of this study was to explore Hu MSC-EXOs whether have a therapeutic effect on POI and provide a kind of new treatments for POI patients.In this study,the commonly used chemotherapy drug cyclophosphamide was used to induce the cell model of POI,and the experiments in vitro were conducted to investigate whether Hu MSC-EXOs has a protective effect on damaged granulosa cells,providing the preliminary experimental basis for the treatment of POI with mesenchymal stem cell exosomes.ObjectiveTo explore whether HuMSC-EXOs has therapeutic effect on POI,and to provide preliminary experimental basis for the treatment of POI with Hu MSC-EXOs.Method1.Primary umbilical cord mesenchymal stem cells were obtained by collagenase digestion for culture and amplification,and the expression of 4th generation Hu MSCs surface factors CD73?CD90?CD105?CD11b?CD19?CD34?CD45 and HLA-DR was analyzed by flow cytometry.2.Collection supernatant of culture solution for Hu MSCs,Hu MSC-EXOs were extracted by differential centrifugation.The concentration of Hu MSC-EXOs protein was quantifed using a BCA Protein Assay Kit.The morphology of Hu MSC-EXOs was observed using Transmission Electron Microscopy(TEM).Hu MSC-EXOs were identifed by specifc antibodies for CD81?HSP70?TSG101 ? Flotillin-1?Calnexin and Lamp 1,which were used for western blot analysis.3.The granulosa cells were divided into three groups,respectively as the negative control group,positive control group and experimental group,and the experimental group was divided into the experimental group 1,2,3,4.Cyclophosphamide 4?g/?l was added to the positive control group and the experimental group,and mesenchymal stem cell exosomes of different concentrations at the same time were added to each experimental group in turn.After mesenchymal stem cells derived exosomes were co-cultured with cyclophosphamide-damaged granulosa cells for 48 h,the apoptosis rate of each group was determined by flow cytometry.Result1.Hu MSCs were adherent to the wall and showed long spindle shape.Hu MSCs surface markers,including CD73?CD90 and CD105,were highly expressed.Furthermore,the negative markers CD11b?CD19?CD34?CD45 and HLA-DR were not expressed,in line with mesenchymal stem cell phenotype.2.Hu MSC-EXOs extracted by ultracentrifugation is deposited in the bottom of the centrifugal tube as a white flocculent by naked eye.The vesicle-like morphology of Hu MSC-EXOs was visualized via TEM,which confirmed exosome diameters of 30 to 200 nm,round or elliptic membranous vesicles with low density electron density.Western blot analysis indicated that Hu MSC-EXOs expressed exosomal markers,such as CD81?HSP70?TSG101 and Flotillin-1 proteins,but did not express the endoplasmic reticulum marker Calnexin or the lysosome marker Lamp 1.3.After HuMSC-EXOs co-culture with cyclophosphamide-damaged granulosa cells in vitro,the apoptosis rate of granulosa cells was significantly reduced.With the increase of exosome concentration,the apoptosis rate of granulosa cells showed a downward trend with dose dependence.ConclusionExosomes derived from human umbilical cord mesenchymal stem cells can reduce the damage of granulosa cells induced by cyclophosphamide in vitro and inhibit the apoptosis of granulosa cells in vitro,which has protective effect on granulosa cells. |
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