Effect of human umbilical cord mesenchymal stem cells on premature ovarian insufficiency in rats
Objective:To investigate the effects and mechanisms of two tail vein transplantation and ovarian in situ transplantation of human umbilical cord mesenchymal stem cells(hUCMSCs)in chemotherapy-induced premature ovarian insufficiency(POI)rat models.Methods:Thirty-two sexually mature female SD rats were randomly divided into four groups with eight rats for each group.No special treatment was given to the blank control group,while the rats in model group,the hUCMSCs tail vein transplantation group(hUCMSCs-iv group)and the hUCMSCs ovarian in situ transplantation group(hUCMSCs-oi group)were treated with 15 days of continuous intraperitoneal injections of cyclophosphamide to establish the POI model.After model successfully established,the rats in the model group were injected with 500 μl of saline in the tail vein,while those in hUCMSCs-iv group treated with a one-time tail vein injection of 1×106 hUCMSCs(resuspended in 500 μl of saline),and those in hUCMSCs-oi group treated with a one-time injection of a suspension of 25 μl of hUCMSCs(resuspended in saline at a concentration of 4×107/ml)from multiple points on the lateral side of ovary tracts.At 14 days after the treatment,ovarian tissues and blood were collected.The body weights,ovarian indices,changes in the estrous cycle,pathological morphological changes in the ovary observed by HE staining,serum estradiol(E2),follicle-stimulating hormone(FSH)and anti-Mullerian hormone(AMH)levels measured by enzyme-linked immunosorbent assay(ELISA),apoptosis-related genes and proteins expression in the ovarian tissues examined by real-time fluorescence PCR and protein immunoblotting,apoptosis of the ovarian cells observed by TUNEL staining,and the follicle-stimulating hormone receptor(FSHR)expression levels in the ovary tissues examined by by immunohistochemical assay were compared among the groups.Results:(1)Compared with those in the blank control group,the majority(87.5％)of rats in the model group had a disrupted pattern of estrous cycles,and the body weight,ovarian wet weight,and ovarian indices were all significantly decreased(P＜0.05).The follicle count was reduced,serum FSH levels were significantly increased while the E2 and AMH levels significantly decreased(P＜0.05)in the model group than that in the blank control group.In the model group,the expression levels of Bax mRNA in ovarian tissue were significantly increased(P＜0.05),while the expression levels of Bcl-2 mRNA and protein significantly decreased(P＜0.05),and the TUNEL-positive rate of ovarian cells was significantly increased(P＜0.05),while the positive expression of FSHR was significantly decreased(P＜0.05).(2)At 14 days after hUCMSCs transplantation,the rate of estrous cycle abnormality in both hUCMSCs treatment groups(hUCMSCs-iv group and hUCMSCs-oi group)was obviously reduce(37.5％,25.0％,respectively)than that in the model group,and the ovarian structure and follicles at all levels were restored to certain degrees compared with those in the model group.The body weights and ovarian indices of the rats in both hUCMSCs treatment groups were significantly increased than those in the model group(P＜0.05).Compared with those in the model group,the serum FSH levels in both hUCMSCs treatment groups were significantly lower(P＜0.05),while the serum E2 and AMH levels were significantly higher(P＜0.05).The results of fluorescence quantitative PCR showed that the relative expression of Bax mRNA in ovarian tissues was significantly reduced while the relative expression of Bcl-2 mRNA was significantly increased in both hUCMSCs treatment groups compared with that in the model group(P＜0.05).Immunoblotting results showed that the expression levels of Bax protein and cleaved caspase-3/caspase-3 in ovarian tissues of both hUCMSCs treatment groups were significantly lower while the expression levels of Bcl-2 protein were significantly higher than those of the model group(P＜0.05).TUNEL staining results showed that the positive rate of TUNEL stained ovarian cells was significantly lower in both hUCMSCs treatment groups than that in the model group(P＜0.05).Immunohistochemistry showed that the expression level of FSHR protein on the membrane of ovarian granulosa cells in both hUCMSCs treatment groups was significantly higher than that in the model group(P＜0.05).Conclusions:In the present study,both intravenous and ovarian in situ transplantation of hUCMSCs were effective in improving ovarian function and follicular structure in chemotherapy-induced POI rats,and this therapeutic effect may be related to the anti-apoptotic effect of hUCMSCs.
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维普
