| PurposesPremature ovarian failure (POF) shows follicular dysfunction. POF caused by chemotherapy has seriously affected the reproductive health of young female cancer patients, and the development of new prevention means imminent. Existing research indicates that POF is closely related to the damage and apoptosis of ovarian granulosa cells (OGCs). Umbilical cord tissue contains large amounts of stem cells with a strong proliferation and pluripotent, known as human umbilical cord mesenchymal stem cells (huMSCs). HuMSCs have all the biological characteristics of mesenchymal stem cells, while its good proliferation and differentiation potential and low immunogenicity, making them as the ideal seed cells for tissue engineering. Many studies have found that huMSCs have used on a variety of tissue injury repairation, like POF, but its mechanism is not entirely clear. The latest study found that exosomes are a class of membranous vesicles 30-200 nm in diameter constitutively released by eukaryotic cells, and they mediate local cell-to-cell communication by transferring micro-RNAs and proteins. HuMSCs synthesize and secrete large amounts of exosomes, which can achieve the purpose of regulating the gene and protein after phagocytosis by target cells. Therefore, this paper examines whether exosomes derived from huMSCs (huMSCs-EXO) would produce protection and repairation as one of the regulatory factors, and uses molecular biology and cytology in vitro to verify functions mentioned above, and deeply explores the molecular mechanisms.MethodsHuMSCs were identified with the expression antigens of cell surface by flow cytometry. OGCs were isolated by trypsin digestion, and identified by immunohistochemistry (anti-FSHR antibody staining). Then the injury model of OGCs induced by chemotherapy drugs (cisplatin) was established. Exosomes were isolated and enriched from stem cells culture supernatant by gradient ultracentrifugation or reagentextraction, observing the shape and size by transmission electron microscope (TEM) and detecting surface markers by Western Blotting. Absorption and phagocytosis of exosomes by cells were observed by fluorescent staining under a confocal microscopy. OGCs were divided into three groups——the control group (A), injury group (B) and co-culture group (C, huMSCs-Exo and OGCs injured by cisplatin were co-cultured), and cultured 48 h under conditions of 37℃, 5% CO2 and 100% H2O, using flow cytometry (Annexin-IV and PI double staining) to detect cell apoptosis rate and Western Blotting to detect the expression of DNA repair proteins, apoptosis proteins, anti-apoptotic proteins (Bcl-2, Bax, Caspase-3, Cleaved Caspase-3, Cleaved PARP, etc.) to verify the protection of exosomes.ResultsHuMSCs were stably amplified by 6-8 generations, which were spindle or polygonal and had morphology liking fibroblasts. HuMSCs expresse matrix receptors (CD44, CD 105) and Integrin marker (CD29), but don’t express hematopoieticlineage markers (CD34, CD45). The rat OGCs purity was 70-80% after FSHR immunohistochemical staining. Exosomes have a double membrane vesicle structure, which diameter is 30-200nm, observed by transmission electron microscopy; and express surface markers such as CD63, CD9, Hsp70, CD81, but don’t conclude Calnexin and Lamp 1. Absorption and phagocytosis of exosomes by cells were observed by fluorescent staining under the microscopy. The ratio of living cells, early apoptotic cells and late apoptotic cells in group A were 85.50±4.45%,5.86±0.50% and 8.69±2.15%, respectively; group B were 71.37±3.10%,8.58±2.04%,17.26± 2.67%; and group C were 80.09±4.00%,5.72±2.15%,10.27±1.46%.The ratio of living cells between A and B, B and C had a significant difference (P<0.05). Western Blotting analysis showed that there were significant differences (P<0.05) between A and B (B and C) about Bax, Cleaved Caspase-3, Bcl-2 and Cleaved PARP. The expression of Bax, Cleaved Caspase-3 and Cleaved PARP in B were increased compared with A, while Bcl-2 was decreased. However, Bax, Cleaved Caspase-3 and Cleaved PARP in C were reduced compared with B, and Bcl-2 was increased.Conclusion and SignificanceExosomes derived from huMSCs have a protective effect on OGCs damaged by chemotherapy in vitro, suggesting huMSCs-EXO is feasible for the prevention and treatment of chemotherapy-induced POF and not only would it provide new targets for POF prevention and treatment, but also provide experience and evidence for studying stem cells function further. This research has not yet reported currently, and it has innovativeness relatively. In addition, stem cells research has good prospects for clinical application, and it is in line with "Translational Medicine" research purposes. |
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