Research On The Effect Of Adipose Stem Cells Derived Exosomes On Premature Ovarian Failure In Rats

Premature ovarian failure?POF?is a clinically heterogeneous disease.With the rapid development of social economy,the incidence rate of POF is on the rise.At present,the main treatment of POF is hormone replacement therapy,which can improve the clinical symptoms of patients,but can not fundamentally improve the ovarian function,so how to provide a safe and effective treatment for POF patients has become a hot spot.A largenumber of studies have confirmed that adipose derived exosomes?ADSCs exo?can play an effective role in the repair of various tissues and organs,but there is no report on the role of ADSCs exo from the same source in premature ovarian failure.In this study,the protective effect of ADSCs exo on ovarian granulosa cells and tissues was studied by using rat granulosa cell injury model and rat premature ovarian failure model,and its mechanism was preliminarily explored,which provided a new safe and effective way for the treatment of POF.Part 1 Isolation and identification of exosomes derived fromadipose stem cells?Objective?Adipose stem cells were extracted from rat inguinal fat by enzyme digestion,and then the exosomes of adipose stem cells were extracted by rapid kit extraction method.Finally,the characteristics of exosomes were identified to verify the reliability of rapid kit extraction method,which laid the foundation for the next step of cell and animal experiments.?Methods and meterials?The adipose tissue of the groin of 12 weeks old rats was cut,and the adipose stem cells were extracted by the method of?-collagenase digestion.The extracted cells were inoculated into the culture bottle for culture.Firstly,the surface markers were identified,and then the ability of multi-directional differentiation of adipogenic,osteogenic andchondrogenic was detected.The third generation ADSCs wich stable passaged were passed down and the cell supernatant was collected.Then exosomes were extracted by rapid kit method.After extraction,electron microscopy detection,particle size analysis and Western Blotting assay of surface markers were performed to complete the identification ofADSCs-EXO.?Result?1.The results of cell morphology showed that the cells grew well and showed atypical fish like appearance.After passage,the cell morphology was stable and showed a long fusiform.2.ADSCs positive markers CD90,CD73 and CD44 were all positive,while CD45,CD34 and CD11b were all negative.The results were consistent with thecharacteristics of the surface markers of adipose mesenchymal stem cells.3.ADSCsobtained in this experiment can be successfully differentiated into adipocytes,osteoblasts and chondrocytes.4.Under transmission electron microscope,ADSCs-EXO has two typical shapes:round or oval,which are lipid bilayers with a diameter of about 50-100nm.5.The results of particle size analysis showed that the median diameter distribution of ADSCs exo was 125.6nm,and the concentration of original particles in suspension was 1.2×10⁹/m L.6.Western blotting showed that CD9,CD63 and TSG101 were positive.?Conclusion?The exosomes can be successfully separated from ADSCs by using the rapid kit method.The ADSCs-EXO can be used in the following experimental studies.Part 2 Establishment of rat premature ovarian failure model induced by?Objective?To verify the method of inducing premature ovarian failure in SD rats by continuous intraperitoneal injection of VCD as reported in the literature,and establish a stable animal model similar to the change process of human premature ovarian failure,so as to lay a solid foundation for follow-up studies.?Methods and meterials?The 1-month-old rats were random Ly divided into blank group,adjuvant group and VCD group for the experiment.The adjuvant group and VCD group were given dailyintraperitoneal continuous injection at the rate of 2m L/Kg?0.2m L/100g?for 15consecutive days.The adjuvant group was injected with sesame oil and the VCD group with VCD working fluid.The estrous cycle was monitored at week 1 after administration,and three rats were random Ly selected at week 4,week 7 and week 10 after administration for orbital blood and bilateral ovarian collection.Serum E₂,FSH and AMH levels in orbital blood were measured by ELISA method,and the changes of morphology,structure and number of follicles in ovary were observed after HE staining.?Result?1.The estrous cycle of rats in the blank group and adjuvant group appeared regularly,and no one of them appeared estrous cycle disorder at the end of the experiment,while all rats in the VCD group had estrous cycle disorder 10th weeks after the completion ofadministration,and all rats were successfully modeled.2.The results of ovarianmorphology observation and follicle count showed that in the blank group and adjuvantgroup,there was no significant change in the ovary,and the number of follicles at all levels was normal;in VCD group,the number of follicles at different stages of development was significantly reduced at the 10th week after administration,the number of primordialfollicles was 20.67±2.52,the number of primary follicles was 26.00±3.00,the number of secondary follicles was 15.00±1.00 and the number of sinus follicles was 9.33±1.533.The serum E₂,FSH and AMH of the blank group and adjuvant group had no significant change at each time point of sampling.At 10w after administration of VCD,serum E₂ and AMH decreased to 29.87±2.54 and 0.76±0.07,respectively,while FSH increased to 20.79±1.32.?Conclusion?The POF model was successfully established by intraperitoneal injection of VCD?160mg/kg?for 15 consecutive days in rats.Part 3 Effects of exosomes derived from adipose stem cells on rat?Objective?To establish and use the VCD induced rat granulosa cell injury model,to explore whether ADSCs-EXO can repair and protect the injured rat granulosa cells,and topreliminarily study its mechanism,so as to lay a foundation for the next animal experiment.?Methods and meterials?Four 3-4-week-old rats were selected to extract ovarian granulosa cells by trypsin method.The proliferation activity and hormone secretion of granulosa cells were further observed.The rat granulosa cells were cultured in the medium containing differentconcentrations of VCD,and the establishment method of the model of granulosa cell injury induced by VCD was determined.The granulosa cells were divided into blank group,VCD group and VCD+ADSCs-EXO group?VCD30?m+ADSCs-EXO10?g?for 48 hours.The inhibition rate,hormone secretion and apoptosis rate of each group were measured.Then the expression of Bcl-2,Bax,Caspase-3,kitl and other related proteins were further detected.?Result?1.The rat granulosa cells extracted by trypsin method have high growth survival rate and stable growth state,which can be used for subsequent experiments.2.The results of Annexin-V/PI double staining showed that the proportion of survival cells in VCD+ADSCs-EXO group was higher than that in VCD group,while the proportion of earlyapoptosis cells and late apoptosis cells was lower than that in VCD group.3.The results of fluorescence quantitative PCR and Western blotting showed that compared with VCDgroup,the expression level of Bax and Caspase-3 in VCD+ADSCs-EXO group was significantly lower,while that of Bcl-2 in VCD+ADSCs-EXO group was significantly higher;compared with VCD group,the expression level of kitl in VCD+ADSCs-EXO group was significantly higher.administration.?Conclusion?ADSCs-EXO can significantly improve and inhibit the stress response and apoptosis of VCD injured granulosa cells,and indeed has a protective effect on granulosa cells.Its mechanism may be that ADSCs-EXO can affect the expression of apoptosis andanti-apoptosis related pathway proteins,so as to achieve the protective effect on granulosa cells.Part 4 Effects of exosomes derived from adipose stem cells on VCDinduced rat premature ovarian failure?Objective?Through the successful model of premature ovarian failure induced by VCD,thispaper discusses whether ADSCs-EXO can repair and protect the ovarian function of rats,and preliminarily studies its mechanism of action,so as to provide a new and effective scheme for the treatment of POF experiment.?Methods and meterials?The rats with normal estrous cycle were divided into blank group and modeling group.After modeling successfully,the rats were divided into blank group,PBS group,ADSCsgroup and EXO group for surgery.In the blank group and PBS group,20?LPBS was injected into each ovary,20?LADSCs cell suspension was injected into each ovary in ADSCs group,and 20?LADSCs-EXO suspension was injected into each ovary in exo group.The estrous cycle was monitored on the first day after operation.The orbital blood and ovaries of each group were collected 28 days after operation.The serum E₂,FSH and AMH levels of orbital blood were measured by the method of ELISA.On the left side of the ovaries to HE staining,follicle morphology count,Tunel staining andimmunohistochemistry,on the right side of the ovary to the Bcl-2,Bax,Caspase 3,KIT,Kitl,PI3K,and AKT related protein expression.?Result?1.The estrus cycle of the blank group appeared regularly,and that of the PBS group appeared irregularly.The estrus cycle of the EXO group and the ADSCs group recovered gradually,but the recovery of the EXO group was faster,the time proportion of the estrus period was better than that of the ADSCs group.2.The number of follicles at all levels in the ovarian tissue of the ADSCs group and the EXO group increased significantly,but the ovarian condition of the EXO group was significantly better than that of the ADSCs group.3.In situ injection of ADSCs and ADSCs-EXO can restore serum E₂,AMH and FSH levels in VCD premature ovarian failure rats,but ADSCs-EXO serum index recovery issignificantly better than ADSCs.4.TUNEL results showed that the apoptotic cells in and out of the follicles in the ADSCs group and the EXO group were significantly reduced,but the apoptotic degree in the EXO group was significantly better than that in the ADSCsgroup.5.The results of immunohistochemistry showed that ADSCs and ADSCs-exo could effectively reduce the expression of Bax and Caspase3,but the effect of ADSCs-exo was better than ADSCs.6.The results of fluorescence quantitative PCR and Western blotting showed that the expression levels of Bax and caspase-3 gene in ADSCs group and EXO group were significantly lower than those in VCD group;the expression levels ofBcl-2,KIT,Kitl,PI3K and Akt gene were significantly higher than those in ADSCs group;however,the decrease and increase of EXO group were significantly better than those in ADSCs group.?Conclusion?ADSCs-EXO can repair and protect the ovarian function of POF rats induced by VCD.The mechanism may be that it can inhibit the apoptosis of ovarian granulosa cells andactivate KIT-KITL-PI3K-AKT signal transduction pathway.ADSCs-EXO has a good clinical transformation and application prospect in the treatment of premature ovarian failure,which provides a new and effective scheme and idea for POF treatment.