| Background: Malignant tumor has always been one of the important reasons that threaten human health.With the improvement of detection methods,the rate of early diagnosis of cancer increases gradually.At the same time,with the improvement of treatment and the rapid development of drug research and development,the survival time of female patients with malignant tumor is gradually prolonged,which makes the impact of chemotherapy on ovarian function increasingly obvious.Follicular reserve is very sensitive to chemotherapy.After chemotherapy,ovarian function can be damaged in the short term,fertility can be reduced in the long term,and the risk of early menopause is significantly increased,such as hot flashes,insomnia,bone calcium loss and so on,which seriously affect the quality of life of patients.Therefore,there are more and more studies on the protection of ovarian function.The existing ovarian protection measures include: cryopreservation of ovarian tissue,cryopreservation of embryos or oocytes,gonadotropin-releasing hormone agonist(Gn RHa)and stem cell therapy.Among them,stem cell therapy is considered to be the most promising treatment at present.Stem cells can constantly renew themselves and have the potential of multi-directional differentiation.Under physiological conditions,it can replace senescent and apoptotic cells,ensuring the integrity of the tissue structure of the body and the exertion of normal physiological functions.Under pathological conditions,it can inhibit inflammatory reaction,promote tissue repair,and contribute to the recovery of damaged tissue structure and function.However,stem cell therapy also has some potential risks,such as vascular embolism caused by transplanted cells,genetic material variation of stem cells,tumorigenesis and teratogenicity,ethical problems and so on.With the discovery of paracrine pathway of stem cells,the research focus has gradually shifted to the exosomes secreted by stem cells.Exosomes not only have the therapeutic effect of stem cells,but also have good flexibility and high cell affinity,and there is no immune rejection and ethical controversy.More and more studies have confirmed that exosomes are involved in many biological processes,including regulation of immune response,maintenance of body balance,blood coagulation,inflammation,angiogenesis and cancer progression.These advantages make stem cell-derived exosomes become a very promising method of acellular therapy,which has been widely concerned in tissue regeneration engineering.In recent years,stem cell-derived exosomes have shown good tissue injury repair function in nervous system,heart disease,lung injury,bone defect,trauma and many other diseases,but there are few studies on ovarian function protection.For this reason,human amniotic fluid mesenchymal stem cells were selected for the first time in this study,and their secreted exosomes were taken as the object of study to explore the role of h AFSc-EXO in ovarian function damage induced by chemotherapy.Objective: To find a suitable isolation method to prepare h AFSc-EXO;to establish the model of ovarian granular(GCs)injury induced by cisplatin in vitro,and to explore the role of h AFSc-EXO in cisplatin-induced ovarian function damage,which lays a foundation for further study of the molecular mechanism of stem cells.Methods: Part 1: Isolation and identification of h AFSc-EXO.1.Amniotic fluid from pregnant women in the second trimester of pregnancy was collected,and h AFSc was purified by cell morphology sorting;2.The stem cell markers on the surface of h AFSc were detected by flow cytometry.The expression of Oct4,Nanog and SOX-2 on the surface of P3 and P8 generation h AFSc was detected by Western blot,and the stemness of different generations of h AFSc was evaluated;3.Conventional ultracentrifugation,concentrated ultracentrifugation and SBI Exosome Isolation Reagent were used to extract h AFSc-EXO,and observed under transmission electron microscope;4.The expression of exosomal enriched protein was detected by Western blot.Part 2: Primary culture of rat ovarian granulosa cells and establishment of cisplatin-induced injury model.1.Pregnant horse serum was used to promote ovulation in small month-old rats,and primary GCs;was extracted;2.The morphology of GCs was observed by crystal violet staining;3.The expression of FSHR on the surface of GCs was identified by immunofluorescence staining;4.The damage degree of GCs induced by different concentrations of cisplatin was observed by crystal violet staining;5.The effect of cisplatin on the proliferation of GCs was evaluated by CCK8,and a stable cell injury model was established in vitro.Part 3: Protective effect of h AFS-EXO on cisplatin-induced GCs damage.According to the different culture conditions and reagents,the subjects were divided into three groups: control group,cisplatin group and h AFSc-EXO group.The cells in the control group were cultured in normal medium and aseptic PBS was added.In cisplatin group,cells were cultured in the medium containing 2.5mg/L cisplatin and aseptic PBS was added.In the h AFSc-EXO group,the cells were cultured in the medium containing 2.5mg/L cisplatin and co-incubated with h AFSc-EXO.1.CCK8 was used to detect the inhibition of GCs proliferation in cisplatin group and h AFSc-EXO group,and the effect of h AFSc-EXO on cisplatin-induced GCs damage was evaluated from the level of cell proliferation;2.TUNEL assay was used to evaluate the effect of h AFSc-EXO on cisplatin-induced GCs injury from the point of view of apoptosis;3.Western blot was used to detect the expression of FSHR,Bax and Bcl-2 in the three groups,and to further evaluate the role of h AFSc-EXO in ovarian function and apoptosis.Results: Part 1: 1.Through the morphological sorting of cells,purified amniotic fluid can be obtained from the second trimester of pregnancy.Microscope observation shows that the h AFSc are slender and spindle-shaped,and the cells are arranged tightly;2.The results of flow cytometry showed that the isolated h AFSc had high stem cell activity and low immunogenicity and could express stem cell markers CD29,CD90,SSEA and Oct3/4,only express major compatibility complex(MHC)class I molecular protein HLA-ABC,but not MHC class ? molecular protein HLA-DR,and does not express hematopoietic stem cell molecular markers CD34 and CD45;3.Western blot results showed that there was no significant difference in the expression of stem cell markers Oct4,Nanog and SOX-2 on the surface of P3 and P8 h AFSc,indicating that h AFSc still had good stem character with the continuous expansion of h AFSc;4.HAFSc-EXO,can be isolated from serum-free exosome culture medium by ultracentrifugation.Under transmission electron microscope,h AFSc-EXO is a disc-shaped vesicle with a diameter of 30-100 nm and has a typical exosome morphology;5.The exosomes isolated and extracted can express exosome-enriched proteins CD9,TSG101 and LAMP1,and h AFSc can also express the same proteins,indicating that the exosomes we isolated from the culture medium are from h AFSc.Part 2: 1.Under microscope,the GCs isolated from rats had the same morphology as that of previous studies,the cells were polygonal or star-shaped,pseudopodia could be seen,and the result of crystal violet staining was the same;2.By immunofluorescence staining,the nucleus was stained blue,and the areas outside the nucleus that could express FSHR were stained red.The results showed that the cells were stained diffusely red,indicating that the FSHR in GCs was highly expressed and the cell viability was good;3.After cultured in normal complete medium for 48 hours,crystal violet staining showed that GCs grew vigorously and cell fusion reached 100%.When cells were treated with 2.5mg/L cisplatin for 48 hours,the number of cells in the pore decreased and some cells necrotic and exfoliated.5mg/L treated with cisplatin for 48 h showed obvious necrosis and exfoliation of GCs;4.CCK8 was used to detect the inhibitory effect of 2.5mg/L and 5mg/L cisplatin on the proliferation of GCs.The results showed that the cell proliferation was inhibited in both groups,and the inhibition of the latter on cell proliferation was more obvious.The 24-hour proliferation inhibition rate of 2.5mg/L cisplatin group was 30.69% ±1.53%,48.86% ±1.93%,54.69% ±1.62%,48.86% ±1.93%,54.69% ±1.62%,48.86% ±1.93%,54.69% ±1.62%,48.86% ±1.93%,54.69% ±1.62%,70.80% ±1.96%.Part 3: 1.2.5mg/L cisplatin treated ovarian granulosa cells for 48 hours,CCK8 detected the cell proliferation inhibition rate of each group.The cell proliferation inhibition rate of h AFSc-EXO group was significantly reduced.The cell proliferation inhibition rate of h AFSc-EXO group was 38.4% ±2.9%,and that of cisplatin group was 49.7%;2.h AFSc-EXO group and cisplatin group were cultured in cisplatin medium containing 2.5mg/L for 48 hours for TUNEL detection.A large number of apoptotic cells were found in cisplatin group,but few apoptotic cells were found in h AFSc-EXO group;3.Western blot showed that compared with the control group,the expression of FSHR and anti-apoptotic protein Bcl-2 decreased and the expression of pro-apoptotic protein Bax increased in cisplatin group and h AFSc-EXO group,indicating that cisplatin caused damage to cell function and increased apoptosis in both groups.Compared with cisplatin group,the expression levels of FSHR and Bcl-2 in h AFSc-EXO group were higher,and the expression level of Bax was lower.h AFSc-EXO reduced the damage of granulosa cells induced by cisplatin and inhibited apoptosis.Conclusion: 1.The h AFSc obtained by cell morphological sorting has strong stemness,low immunogenicity and no hematopoietic stem cell components;2.The h AFSc-EXO content obtained by conventional ultracentrifugation is higher and the impurities are less;3.2.5 mg/L cisplatin can establish a stable model of GCs injury in vitro;4.h AFSc-EXO can reduce the apoptosis of GCs induced by cisplatin,reduce the inhibitory effect of cisplatin on the proliferation of GCs,and improve ovarian function. |
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