| Objective: To study the repair effect and the mechanism of adipose mesenchymal stem cells on epirubicin-induced premature ovarian failure in mice.Methods:Twenty five 5-week-old female mice with normal estrous cycle were randomly divided into blank control group,model group(POF group)and treatment group;model group and treatment group were intraperitoneally injected with epirubicin 10 mg / kg twice on the 1st and 3rd day,and the changes of estrous cycle were observed by vaginal smear every day.Ten days after successful modeling,m ADSCs cells were injected into the treatment group and divided into three groups: tail vein injection of m ADSCs group(m ADSC1),ovarian in situ injection of m ADSCs group(m ADSC2),and ovarian in situ injection of m ADSCs combined with 75ug/kgb FGF group(m ADSC3).The blood and ovarian tissues of each group were taken 7 days after m ADSCs cell treatment;the levels of estradiol(E2),anti-Mullerian tube hormone(AMH)and follicle stimulating hormone(FSH)in the serum of each group were detected by Elisa method;the paraffin sections of the left ovarian tissue of each group were prepared,and the number of follicles was counted by HE staining;the fluorescence intensity of the staining results was analyzed by TUNEL staining combined with Image J software.Total RNA and protein were extracted from the right ovary,and the expression levels of germ cell related genes(Gdf9,Figla,Gpd1,lcp2,Rbm24),apoptosis related genes(Bax,Casepase3,Casepase9),anti-apoptosis related genes(Bcl2)and granulosa cell specific genes(Foxl2,Inhibin)in the ovarian tissues of mice in each group were detected by q PCR and Western blotting,and the related data were statistically analyzed.Results:(1)The estrous cycle of mice in Control group was regular.The estrous cycle of POF group and treatment group was disordered 2 days after injection of epirubicin,and the CIPOF mouse model was successfully established.(2)The results of serum hormone detection showed that compared with Control group,the levels of AMH and E2 in POF group decreased by 3.52±0.11 vs 5.76±0.27,P< 0.05;209.12±5.85 vs 260.09±16.55,P< 0.05;the level of FSH increased by 37.42±1.38 vs28.44±0.87,P< 0.05,the differences were statistically significant.the values of E2 in m ADSC1,m ADSC2 and m ADSC3 treatment group were 272.16±12.05,258.39±9.04,300.25±29.34 respectively.The AMH value in each treatment group were 5.64±0.14,6.00±0.71,6.11±0.13 respectively.which were higher than those in POF group,and the difference was statistically significant(P< 0.05).The FSH values were 29.17±1.56,26.84±2.47 and 30.23±1.03 respectively,which were lower than those in POF group,and there was significant difference(P < 0.05).There was no significant difference between the treatment group and the Control group(P >0.05).(3)The results of follicular count in ovarian tissues of each group showed that:In the Control group,the number of primordial follicles was 101.67±4.51,the number of primary follicles was 78.00±4.58,the number of secondary follicles was 75.33±3.52 and the number of atresia follicles was 32.33±1.15.In the POF group,the number of primordial follicles,primary follicles,secondary follicles and atresia follicles was 79.33±5.51,56.67±3.51,58.67±3.51 and45.00±2.00,respectively.There were significant differences in the number of follicles at all levels between the two groups(P <0.05).At the same time,the blood vessels of ovarian interstitium decreased and the cortex thinned in POF group under microscope.The number of primary and secondary follicles in m ADSC1 and m ADSC2 groups were higher than that in the POF group,and the number of atretic follicles was lower than that in the POF group,with significant differences(P<0.05).The number of primordial follicles was higher than that in the POF group,but there was no statistical significance.Compared with the POF group,the number of primordial,primary and secondary follicles in m ADSC3 group increased,and the number of atresia follicles decreased,with statistical significance(P<0.05).(4)TUNEL staining results showed that: compared with the Control group,the number of apoptotic cells in the ovarian tissue of the POF group increased,and most of the apoptotic cells were granulosa cells.The apoptotic cells were significantly reduced in the ovarian tissues of m ADSC1,m ADSC2 and m ADSC3 groups.(5)The results of q RT-PCR showed that:compared with the control group,the expression of germ cell-related genes Inhibin,Gdf9,Figla,Gpd1,lcp2,Rbm24,Foxl2 and anti-apoptotic gene Bcl2 in POF group decreased significantly,while the expression of pro-apoptotic genes Bax,Casepase3 and Casepase9 increased significantly;the expression of germ cell-related genes in m ADSC1,m ADSC2 and m ADSC3 treatment group was higher than that in POF group;the expression of pro-apoptotic gene was significantly lower than that in POF group,but still higher than that in control group.(6)The result of Wester blotting protein hybridization showed that:the expression levels of Caspase-3,Caspase-9 and Bax in POF group were increased compared with Control group,while the expression levels of Caspase-3,Caspase-9 and Bax in m ADSC1,m ADSC2 and m ADSC3 groups were decreased compared with POF group,and the expression level of stem cell in situ treatment group(m ADSC2)was the closest to that in Control group.Conclusion:(1)The mouse model of premature ovarian failure induced by epirubicin was successfully established.(2)Adipose mesenchymal stem cell transplantation can effectively repair the ovarian function injury caused by epirubicin chemotherapy.(3)The mechanism of m ADSCs in the treatment of premature ovarian failure may be through promoting the growth and development of primordial follicles and inhibiting the apoptosis of granulosa cells. |
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