Premature ovarian failure (POF) has a great impact on reproductive endocrine function in females, and it is an important cause of infertility. Previous studies have demonstrated that small extracellular vesicles (sEVs) derived from stem cells play an important role in tissue regeneration. This study aimed to investigate the therapeutic effect of sEVs derived from embryonic stem cells (ESCs-sEVs) on damaged ovaries and explore the underlying molecular mechanisms. Mice POF models were established by injecting mice with cyclophosphamide and busulfan. Then, ESCssEVs were intravenously transplanted into POF mice. The plasma of mice was harvested at 1 and 2 weeks after treatment to analyze the levels of anti-Mullerian hormone (AMH), estradiol (E2), and follicle stimulating hormone (FSH) by ELISA. The morphology of ovaries and follicles was observed by H&E staining, and apoptosis of granulosa cells was detected by TUNEL. In vitro, EdU and CCK-8 tests were used to evaluate the proliferation of cultured granulosa cells stimulated by ESCs-sEVs. Western blotting was used to determine the expression of PI3K/AKT and apoptotic-related proteins. After transplantation of ESCs-sEVs, the levels of serum sex hormones recovered to normal levels. In addition, the number of follicles was significantly increased, and the number of apoptotic cells was decreased. The results in vitro revealed that ESCs-sEVs could significantly improve the proliferation rate of granulosa cells and increase the expression of phosphorylated PI3K and AKT. Meanwhile, the positive effect on proliferation and the negative effect on apoptosis observed in granulosa cells were obviously decreased when the PI3K/AKT signaling pathway was inhibited. Our findings suggested that ESCs-sEVs could improve ovarian function by regulating the PI3K/AKT signaling pathway, which could provide a promising clinical therapy for POF.
L'insufficienza ovarica precoce (POF) ha un grande impatto sulla funzione endocrina riproduttiva nelle femmine, ed è una causa importante di infertilità. Precedenti studi hanno dimostrato che piccole vescicole extracellulari (sEVs) derivanti da cellule staminali svolgono un ruolo importante nella rigenerazione dei tessuti. Questo studio mira a indagare l’effetto terapeutico degli sEVs derivati da cellule staminali embrionali (ESCs-sEVs) sulle ovaie danneggiate e ad esplorarne i meccanismi molecolari. I modelli di POF di topi sono stati stabiliti iniettando ciclofosfamide e busulfano nei topi esaminati. Poi, ESCs-sEVs sono state trapiantate per via endovenosa in topi POF. Il plasma dei topi è stato raccolto 1 e 2 settimane dopo il trattamento per analizzare i livelli dell’ormone anti-mulleriano (AMH), estradiolo (E2), e dell’ormone follicolo stimolante(FSH) con ELISA. La morfologia delle ovaie e follicoli è stata osservata dalla colorazione H&E, e l’apoptosi delle cellule della granulosa sono state rilevate dal saggio del TUNEL. In vitro, il test Edu e CCK-8 test sono stati utilizzati per valutare la proliferazione delle colture granulosa stimolata da ESCs-sEVs. Infine il Western blotting è stato utilizzato per determinare l'espressione di PI3K/ AKT e delle proteine apoptotiche-correlate. Dopo il trapianto di ESCs-sEVs, i livelli sierici degli ormoni sessuali hanno ripristinato i valori normali. Inoltre, il numero di follicoli è significativamente aumentato e il numero di cellule apoptotiche è diminuito. I risultati in vitro hanno rivelato che ESCs-sEVs potrebbero migliorare significativamente il tasso di proliferazione delle cellule della granulosa e aumentare l'espressione di PI3K e AKT fosforilato. Al contrario, l'effetto positivo sulla proliferazione e gli effetti negativi sull'apoptosi osservati nelle cellule della granulosa sono diminuiti quando il segnale della via PI3K/AKT è stato inibito. I risultati suggeriscono che ESCs-sEVs potrebbero migliorare la funzione ovarica regolando il percorso di segnalazione PI3K/AKT, assicurando una promettente terapia clinica per POF.
Insufficienza ovarica precoce: ripristino delle funzioni attraverso l'attivazione della via di segnalazione PI3K/AKT
ROBBE, GIOVANNA ALESSIA
2019/2020
Abstract
Premature ovarian failure (POF) has a great impact on reproductive endocrine function in females, and it is an important cause of infertility. Previous studies have demonstrated that small extracellular vesicles (sEVs) derived from stem cells play an important role in tissue regeneration. This study aimed to investigate the therapeutic effect of sEVs derived from embryonic stem cells (ESCs-sEVs) on damaged ovaries and explore the underlying molecular mechanisms. Mice POF models were established by injecting mice with cyclophosphamide and busulfan. Then, ESCssEVs were intravenously transplanted into POF mice. The plasma of mice was harvested at 1 and 2 weeks after treatment to analyze the levels of anti-Mullerian hormone (AMH), estradiol (E2), and follicle stimulating hormone (FSH) by ELISA. The morphology of ovaries and follicles was observed by H&E staining, and apoptosis of granulosa cells was detected by TUNEL. In vitro, EdU and CCK-8 tests were used to evaluate the proliferation of cultured granulosa cells stimulated by ESCs-sEVs. Western blotting was used to determine the expression of PI3K/AKT and apoptotic-related proteins. After transplantation of ESCs-sEVs, the levels of serum sex hormones recovered to normal levels. In addition, the number of follicles was significantly increased, and the number of apoptotic cells was decreased. The results in vitro revealed that ESCs-sEVs could significantly improve the proliferation rate of granulosa cells and increase the expression of phosphorylated PI3K and AKT. Meanwhile, the positive effect on proliferation and the negative effect on apoptosis observed in granulosa cells were obviously decreased when the PI3K/AKT signaling pathway was inhibited. Our findings suggested that ESCs-sEVs could improve ovarian function by regulating the PI3K/AKT signaling pathway, which could provide a promising clinical therapy for POF.| File | Dimensione | Formato | |
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Tesi Giovanna Alessia Robbe.pdf
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https://hdl.handle.net/20.500.12075/3461