| ObjectiveTo investigate the repairing effect and mechanism of bone marrow mesenchymal stem cells(BMSCs)and their derived exosome(EXO)on chemotherapy-induced premature ovarian failure(POF).MethodsBMSCs were isolated,cultured and identified in vitro.Exosomes from BMSCs were extracted and identified.After establishing POF rat model,BSMCs and their derived EXO were transplanted to treat POF rats.The methods are as follows to evaluate the therapeutic effects of BMSCs and exo on POF rat model:(1)Evaluating the general situation of rats: body weight,spirit,hair,eating and defecation(2)Detecting the estrous cycle by vaginal smear(3)Detecting serum hormones by ELISA(4)Counting ovarian follicles by HE staining(5)Evaluating tissue apoptosis and the expressions of apoptotic protein by Tunnel and immunohistochemistry.Ovarian granulosa cells(GCS)of rats was cultured and identified in vitro.GCs damaged by cyclophosphamide(CTX-GCs)was used to establish a vitro POF model.A vitro POF model(CTX-GCs)was established,and the co-culture system of CTXGCs with BMSCs and their derived EXO was established.The apoptosis of GCs were dected by AnnexinV/PI to assesse the therapeutic effect of BMSCs and their derived EXO on CTX-GCs apoptosis.The uptake of BMSCs-derived EXO by CTX-GCs were observed and the expression of mir-144-5p were dected by qRT-PCR.After the change of mir-144-5p level in BMSCs-derived EXO in the co-culture system,the expression of PTEN as miR-144-5p downstream targeting gene was detect Results 1.The therapeutic effect of BMSCs and their derived exosomes on POF: BMSCs had a uniform morphologica appearance as fibroblast-like long spindles in an ordered arrangement.BMSCs showed positive expression of CD90 and CD105,and negative expression of CD34,CD19 and CD45,which was consistent with BMSCs phenotype.The characteristics of BMSC-derived exosomes were analyzed by electron microscopy,which revealed vesicular structure with a diameter of 30-100 nm.The expression of the exosomal marker proteins CD9 and CD81 was identified circulating exosomes.Rats in the POF group appeared body weight loss,decreased activity,body hair dull and decreased in eating.Meanwhile,irregular estrus cycle,decreased E2 and AMH,increased FSH and LH in the serum,increased atresia follicles,increased GCs apoptosis,and increased apoptotic protein expression were also observed in POF group.Comparaed with PBS group,the improved general situation,restored the estrus cycle,increased the basal follicles and sinus follicles while decreased atresia follicles,increased E2 and AMH and decreased FSH and LH levels in the serum were observed in the BMSCs group or the EXO group.The morphological analysis of the GCs revealed polygonal and fibrous-like structures GCs with positive expression of FSHR.BMSCs-derived exosomes could be transferred to the co-cultivated CTX-damaged GCs,leading to GCs apoptosis inhibition.The apoptosis rate of CTX-damaged GCs cocultured with BMSCs or BMSC-derived exosomes was decreased.However,when GW4869 prevented the release of exosomes BMSCs,the apoptosis rate of GCs did not decrease.2.Mechanism of BMSCs and their derived exosomes in repairing POF: After the treatment with BMSCs and their derived EXO,the expression of mir-144-5p was increased while the expression of PTEN was decreased in ovarian tissue of POF rats.The expression of miR-144-5p expression was increased while the expression of PTEN was decreased in CTX-GCs cocultured with BMSCs or EXO.When overexpression or inhibition of miR-144-5p in BMSCs-derived EXO in the co-culture system,the expression of PTEN was corresponding decreased or increased,,accompanied with increased or decreased p-AKT.ConclusionBMSCs inhibit the apoptosis of GCs and mediate the repair of ovarian function by transmiting exsomal mir-144-5p into GCs to inactivated the PI3K/AKT pathway by suppressing PTEN targeting... |
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