Experimental Study Of Human Umbilical Cord Mesenchymal Stem Cells In Premature Ovarian Failure Therapy

Background Premature ovarian failure(POF)is a disease characterized by amenorrhea,infertility,estrogen deficiency and elevated gonadotropin levels before the age of 40.Chemotherapy is an effective method for the treatment of malignant tumors,but chemotherapy drugs can cause different degrees of damage to the patient's ovaries,and some patients even develop into premature ovarian failure.Cyclophosphamide(CTX)is a commonly used alkylating agent in chemotherapy.It is of great significance to study the treatment of POF caused by CTX.At present the treatment of POF schemes can not repair the ovarian function fundamentally,in recent years mesenchymal stem cells for the treatment of POF has become a research hotspot.Human umbilical cord mesenchymal stem cells(hUMSCs)stem from the abandoned umbilical cord,which is resouceful and obtained conveniently.hUMSCs,pluripotent stem cells,belonging to the immunodeficiency cells becomes the key research object for POF.Objects(1)hUMSCs was isolated by tissue explants adherent method and its biological characteristics were identified.(2)Observing the ability of hUMSCs to differentiate into adipogenic,osteogenic and chondrogenic.(3)To evaluate whether the animal model of POF induced by CTX is successful or not.(4)To explore the therapeutic effect of hUMSCs on POF rats.(5)To explore the possible mechanisms of hUMSCs repairing ovarian function.Methods The hUMSCs was isolated from umbilical cord and cultured with the tissue explants adherent method.Cell morphology was observed under microscope;flow cytometry identified cell surface antigens;inducing to differentiate into adipogenic,osteogenic and chondrogenic,observing its multidifferentiation.POF model was established by intraperitoneal injection of cyclophosphamide in SD rats.POF rates were injected into 10mg/ml CTX on the 1st day and 3.2mg/ml CTX from the2 nd to 15 th day,one time per day,5m L/kg.The general situation of rats was observed and recorded.Combining with the detected content of follicle stimulating hormone(FSH)and estradiol(E2)in scrum,ovarian morphology and changes of follicular to judge whether the POF model is successful or not.The tail vein of each treatment group rat was injected with 1×106/ml hUMSCs,and the distribution and differentiation of hUMSCs in the ovary were detected by fluorescence in situ hybridization(Fluorescence in situ,FISH).After the transplantation of hUMSCs,the content of follicle stimulating hormone(FSH),luteinizing hormone(LH),serum inhibin inhibin B(INHB)and anti mullerian hormone(AMH)in Serum were tested to evaluated improvement on ovarian reserve function.Caspase-3 signal expression was detected by immunohistochemical.Morphology of ovarian tissue and the number of follicles were observed after HE staining.Ovarian ultrastructure changes were observed by transmission electron microscopy.The distribution and differentiation of hUMSCs were detected by FISH.Possible mechanism that hUMSCs cures POF is speculated which is based on above detection results.14 days after pregnant,the rats were dissected to observe the numberof live and absorbed embryos and compare the fertility of three groups.Results(1)The cells isolated from the umbilical cord were observed under the microscope,and most of them were spindle shaped.The high expression of CD73,CD90 and CD105 and low expression of CD34,CD45,CD19,CD11 b,HLA-DR were detected by flow cytometry.The surface antigen and cell morphology were detected,showing that the cells were hUMSCs.(2)After oil red O staining,the cell's change in shape from long fusiform to oval or round and dyed red light lipid droplets in cells were observed,showing success of adipocyte differentiation.After alizarin red staining,the cell's change in shape from long fusiform to polygon and calcium salt deposition were observed,suggesting that the the success of osteoblast differentiation.After alcian blue staining,dyeing cells were strongly positive,however cells without differentiation medium were loose and cells' morphology was fusiform,suggesting that the success of chondrogenic differentiation.It is proved that hUMSCs has the potential of multi-directional differentiation.(3)Compared with the negative control group,E2 in serum of rats in model group decreased and the content of FSH increased.HE staining in the rats of model group showed that the number of growing follicles decreased and atretic follicles increased,according with typical characteristics of clinical POF,which showed that the POF model was successfully established.(4)17 days after hUMSCs transplantation,the serum levels of INHB and AMH in the hUMSCs transplantation group were higher than those in the model group.HE staining showed that the number of growing follicles in transplantation group increased and atretic follicles decreased on the 1st and 17 th days,which indicated that hUMSCs could improve the ovarian reserve and repair the ovary.(5)The results of FISH showed that hUMSCs was distributed in the ovarian tissue,and a few differentiated into ovarian granulosa cells and oocytes.Electron microscopy showed that the granulosa cells of hUMSCs group were gradually repaired.Caspase-3 positive signal was not detected in the growing follicles of hUMSCs transplantation group,and the expression of Caspase-3 was lower than that of model control group.It is speculated thathUMSCs may repair the ovary by repairing granulosa cells to inhibit the growth of follicular apoptosis and differentiating into oocyte and granulosa cells.(6)There was an increasing trend in the number of live embryos in hUMSCs transplantation group,but there was no significant difference between model group and control group.Conclusions(1)hUMSCs can be successfully isolated and cultured by tissue adherent method.HUMSCs is long spindle shape and has multidifferentiation,which is similar to that of mesenchymal stem cells.(2)The rat model of POF was established successfully.HUMSCs can improve the ovarian reserve function of POF rats and repair the ovary.(3)hUMSCs may repair the ovary by repairing the granulosa cells of the ovary,inhibiting the growth of follicle apoptosis,partly differentiating into germ cells and granulosa cells.(4)hUMSCs could improve the fertility of POF rats.