Effect And Mechanism Of UCMSC On Ovarian Structure And Function In Natural Aging C57 Mice

Objectives:1.The natural ovarian aging model of C57 mouse was screened and obtained to provide experimental objects for therapeutic research.2.The preparation of C57 mouse umbilical cord mesenchymal stem cells(mUCMSC)provides materials for therapeutic research3.To evaluate the therapeutic effect and mechanism of mUCMSC on aging C57 mouse model and explore the mechanism that mUCMSC promotes antioxidant repair in mouse granulosa cells(mGCs)Methods:1.Screening and Evaluation of Ovarian Aging Model of C57 Mouse:50 female C57 mice of 8 days and 8 months were purchased from Weitonglihua.The weight are(15±2.5)g and(20±5)g,and the regular feed was fed until 2 months and 18 months respectively.Three mice were taken and observed via vaginal smears for 30 days.The level of hormone E2,FSH,AMH and INH-B were tested.The ovarian tissue was embedded in paraffin sections.The ovarian structure was observed by HE staining and the antral follicles were counted2.Preparation and identification of mUCMSC:8 C57 pregnant rats of on the 18th day of gestation were selected and the umbilical cord was collected under sterile condition in vitro.The umbilical cord was cultured by tissue mass attachment culture and purified by trypsin digestion.The morphology was observed under the microscope.The proportion of CD29,CD34 and CD90 positive cells was analyzed by flow cytometry.The differentiation of mUCMSC into bone,fat and chondrocytes was induced and the differentiation potential was analyzed.3.mUCMSC transplantation:18-month-old C57 mice were randomly divided into model control group and treatment group.At the same time,two-month-old C57 mice were set as young control group(15 mice in each group).mUCMSC,1×107/kg,labeled with GFP was injected into the tail vein of the treatment group.The model control group and the young control group were injected with the same volume of saline for 3 weeks(every Monday and Thursday).One month after the last injection of mUCMSC,the therapeutic effect was evaluated and the mechanism of the treatment was studied.4.The evaluation of curative effect of mUCMSC transplantation:the hair color of mice was observed and the ovary index was calculated;the levels of serum hormone E2,FSH,AMH and INH-B were detected by ELISA;the morphology and size of ovary were observed by gross anatomy;the ovary tissue structure was observed by HE staining and the antral follicles were counted.5.Study on the mechanism of action of mUCMSC:(1)Immunohistochemistry analysis of expression level of ovarian senescence-related protein(P53,P16,SOD1),autophagy-associated protein(becline1,LC3b,sirt1,sirt3,p62),apoptosis-related protein(Bar,Bcl-2,Caspase-3)and granulosa cell specific protein FSHR;(2)The apoptosis of ovarian tissue was observed by Tunel staining.(3)The transcription level of aging-related genes(P53,SOD 2),autophagy-associated genes(beclinel.LC3b,sirt1),apoptosis-related genes(Bax,Bcl-2,Caspase-3)and peroxidase genes(PRD.Y ?,PRDX ?,PRDX ?,PRD ?)were detected by qRT-PCR:(4)sequencing of mouse ovarian granulosa cell transcript group:Smart-seq2 technique was used to sequence the mRNA of C57 mouse ovarian granulosa cells,and bioinformatics method was used to analyze the sequencing data.The transcription level of granulosa cells in the three groups was observed.(5)The primary mGCs of C57 mice was cultured to P1 generation and induced by H2O2 of 1mmol/L for 4 hours to establish the oxidative stress model of mGCs.The model was co-cultured with mUCMSC indirectly with Transwell plate.The flow cytometry was used for the detection of apoptosis and ROS level on mGCs.qRT-PCR was used to detect the transcription level of apoptosis-related gene Bax and anti-apoptosis gene Bcl-2.Results:1.Compared with young C57 mice,the estrous cycle of aged C57 mice(18 months old)was disordered and the]evel of serum INH-B was decreased.The ovarian volume became smaller and ovarian parenchyma was occupied by interstitial cells and follicles disappeared.2.The mUCMSC:The mUCMSC cultured by tissue block adherent method crawled out the next day and the fusion rate reached 80%on the 6th day.The mUCMSC was passaged to the P3 generation with a spindle shape.The positive rate of positive markers CD90 and CD29 was 100%and 98.6%,and the positive rate of negative markers CD34 was 0.39%.Saturated oil red O staining was positive in P4 generation mUCMSC after lipogenic induction differentiation.Alizarin red staining was positive after osteogenic induction differentiation.Arnephrine staining was positive after chondrogenic induction differentiation.3.The curative effect of mUCMSC transplantation:compared with the model control group,the hair color of the treatment group became black and bright and the white hair disappeared,and the ovary index of the mice was higher than that of the model control group.The levels of serum E2,AMH,INH and B in mice were significantly higher than those in model control group(p<0.05).In the treatment group,the gross observation volume of ovary became larger,and the histologic observation showed that the ovary structure was clear,the follicles of all grades and antral follicles were increased in the cortex of the treated group.4.The results of mUCMSC mechanism study:(1)the results of immunohistochemistry showed that the aging-related genes(P16,SOD1)and autophagy-associated genes(becline1,LC3b)in the treatment group were significantly higher than those in the model control group.The protein level of apoptosis gene Caspase-3 and granulosa cell-specific expression gene FSHR decreased,and the expression level of autophagy-associated gene sirt3 increased(p<0.05).(2)Tunel staining showed that the apoptosis rate in the treatment group was significantly lower than that in the model control group(p<0.05).(3)The results of qRT-PCR showed that the transcription level of apoptosis-related gene(Bax.Caspase-3)and autophagy-associated gene(LC3b)were decreased in the treatment group(p<0.05).The transcription level of senescence-associated gene(SOD2)and peroxidase gene(PRDX ?,PRDX ?)were increased(p<0.05).(4)Sequencing results:Compared with the young control group,the model control group was up-regulated in the genes involved in the inflammatory response and immune response,and involved in the PI3K-Akt signaling pathway,the P53 signaling pathway,the regulation of cell population proliferation,and the down-regulation of IGFBPs regulation of IGF transport and uptake regulation.In addition to the down-regulation of genes involved in the inflammatory response,the other treatments are up-regulated;(5)In the model of mGCs oxidative stress,compared with the control group,the cell apoptosis increased,the ROS level increased,the transcription level of apoptotic gene Bax increased and the transcription level of anti-apoptotic gene Bcl-2 decreased in the model group.After co-culture with mUCMSC,the cell apoptosis decreased,the ROS level decreased,the transcription level of apoptotic gene Bax decreased and the transcription level of anti-apoptotic gene Bcl-2 increased(p<0.05).Conclusions:1.C57 mice that were routinely raised to 18 months had smaller ovarian volume,decreased INH-B levels,and disappeared follicles at various levels,which could be used as a model for studying ovarian aging.2.mUCMSC with high purity,high value-added activity and multi-directional differentiation potential was obtained by tissue block adhesion method.3.After the transplantation of mUCMSC for the treatment of ovarian aging mice,the ovarian volume of the mice increased,follicles at all levels were observed in the cortex,the count of antral follicles increased,and serum E2,AMH and INH-B levels increased,indicating that the ovarian reserve function was significantly improved,and mUCMSC had a curative effect on ovarian aging.4.mUCMSC down-regulates the expression of apoptosis-related genes(Bax,Caspase-3),and up-regulates the expression of SOD2.peroxidase gene PRDX ?,and simultaneously reduces the apoptotic rate and ROS level of granulosa cells,and also regulates immunity and anti-inflammatory.It also up-regulates PI3K-Akt and P53 signaling pathways,up-regulates cell population proliferation,and IGFBPs regulate gene expression of IGF transport and uptake to promote the repair of senile ovaries.5.The oxidative stress model of mGCs was established successfully.Co-culture of mUCMSC could improve the antioxidant stress ability of mGCs and decrease the apoptosis of mGCs.