The Reconstruction Effect Of MSC-MVs On Ovarian Function Of Chemotherapy Induced Premature Ovarian Failure In Mouse Model

Part I Isolation and purification of mesenchymal stem cell-derived microvesiclesObjective To establish an effective method for isolating and purifying mesenchymal stem cell-derived microvesicles.Methods Mesenchymal stem cells(MSCs)were isolated from human umbilical cord Wharton's jelly through explant culture method.Passage 3 to 7 of MSCs cultured under serum-deprivation condition.Then supernatant were collected and MVs in supernatant were extracted and purified through the methods of modified ultracentrifugation,ultracentrifugation and sucrose gradient ultracentrifugation.The morphology of MVs were observed under electron microscope and protein quantification of MVs was carried out with the method of Bradford.Results(1)MSC isolation and culture MSCs showed an polygonal-like shape after 6-7 days culture,and reached 80% confluence and presented predominantly spindle-shape morphology after 17-20 days culture.(2)Isolation of MSC-MVs and morphological observation MSC-MVs characterized as a round-shaped membrane vesicles of approximately 30 to 200 nm in diameter under electron microscope,and the protein content of MVs released by 106 MSCs was approximately 162.66±17.50?g through the modified ultracentrifugation.Conclusion High quantity of MVs could be obtained by the modified ultracentrifugation method.Part? The location of MVs in mouse ovary after transplantationObjective To investigate the location of MVs derived from UC-MSC in mouse ovary.Methods PKH26 labeled MVs were injected into normal mice and POF model via the tail vein.After 12 h,24h,48 h,72h,and 1 week,the ovaries were taken,and the frozen sections were staining with FSH-R,nuclei were stained with DAPI.The localization of PKH26-labeled MVs in ovaries were observed under confocal laser scanning microscope.Results PKH26 labeled MVs were detected in theca layer of ovarian follicle in CTX-mice12 h after MVs were transplanted.The red fluorescence signal of MVs in CTX-mice was stronger 24 h after transplantation.Further more,the signal reached its maximum 48 h after transplantation and incorporated into the cytoplasm of granulosa cell(GC)both in CTX-mice and normal mice.The labeled signals almost disappeared 1 week later.Conclusion MVs could locate around follicle and absorb by GCs in normal mice as well as CTX-mice after injecting via tail vein.Part? The effect and mechanism of MSC-MVs on ovarian function of chemotherapy induced premature ovarian failure in mouse modelObjective To investigate the effect and mechanism of MSC-MVs on ovarian function of chemotherapy induced premature ovarian failure mouse model.Methods The mice were divided into 3 groups: normal control group(group 1,Non-PBS-mice),chemotherapy-induced POF group(group 2,CTx-PBS-mice)and MVs treatment group(group 3,CTx-MVs-mice).Mouse model of premature ovarian failure was established by administration of cyclophosphamide(CTX)200mg/kg and busulfan(BUS)20mg/kg through intraperitoneal injection.After POF models were built,MVs were injected into each experimental model via tail vein in group 3.A equivalent volume of PBS were injected into those in group 1 and group 2 respectively.The operation was repeated once a week for 4 times in total.Vaginals mears cell smears were observed daily.Mice were weighted once a week,and executed at 4 weeks after MVs or PBS administration.Ovarian function were assessed via follicle counts,levels of FSH and E2,blood vessel counts,CD34 positive cells in corpus lutea(CL),angiogenesis-related m RNA and protein expression of ovarian.Results:(1)Establishment of Premature Ovarian Failure Mouse Model Cyclicity of vaginals mears was irregular in chemoablated animals.Besides,Oocyte degeneration,severe interstitial fibrosis,significant follicles reduction(p<0.05),increased atretic follicle propotion(p<0.05)was observed in CTx-mice ovarian tissue.The levels of E2 and FSH in CTx-mice were makedly changed(p<0.05).(2)Basic characteristics of mice Following appearance of anorexia,fatigue,lethargy,and irregular oestrous cycles,the weight of mice in group2 decreased significantly compared with those in group 1(p<0.05).After MVs transplantation,the weight of mice in CTX-MVs-mice group was significantly increased(p<0.05)following the improvement of feeding and activity.Besides,regular oestrous cycles were restored in 75% subjects 4 weeks later.(3)Histology of ovaries More primordial,developing and preovulatory follicles were observed in CTx-MVs-mice than those in CTx-PBS-mice(p<0.05).(4)The levels of FSH and E2 Four weeks after MVs transplantation,the level of serum E2 in the CTX-MVs-mice was significantly higher and FSH level was markedly lower compared with CTX-PBS-mice group(p<0.05).However,the sex hormone levels did not recovery to normal compared with Non-PBS-mice group(p<0.05)(5)Blood vessel and CD34-positive cell Counts The number of blood vessels and CD34-positive cells in CTX-MVs-mice group increased significantly 4 weeks after MVs injection compared with CTx-PBS-mice group(p<0.05),which were still low compared with Non-PBS-mice group(p<0.05).(6)Vascular m RNA expression in ovarian tissue Results showed that a significant reduction in m RNA expression of vascular endothelial cell growth factor(VEGF),insulin-like growth factor-1(IGF-1)and angiogenin in CTx-PBS-mice group compared with Non-PBS-mice group(p<0.05).However,following MVs transplantation,the m RNA level was significantly increased compare with the CTx-PBS-mice group(p<0.05).(7)Vascular protein expression in ovarian tissue Compared with CTx-PBS-mice group,the protein expression of VEGF,Akt and p-Akt were increased significantly in CTX-MVs-mice group(p<0.05).Conclusion MVs transplantation could partly relieve the damage of ovary caused by chemotherapy,and the probable mechanism might be that MVs could promote angiogenesis of injured ovarian tissue and improve the microenvironment of ovarian.