Investigation On Treatment Of Human Umbilical Cord Mesenchymal Stem Cells In Autoimmune Premature Ovarian Failure Of Rats

Premature ovarian failure（POF）refers to the phenomenon that women have amenorrhea,decreased estrogen levels,increased gonadotropin levels,and ovarian atrophy before the age of 40.POF is one of the main causes of female infertility.The purpose of our current experiments is to develop an autoimmune rat POF model,and examine effects of human umbilical cord mesenchymal stem cells（h UCMSCs）on rat POF.By comparing h UCMSCs transplantation methods and transplant doses to judge the treatment effects.At the same time,the potential mechanism of h UCMSCs in the treatment of POF was preliminarily explored.This experimental study included four sections.1.Establishment of autoimmune rat POF modelObjective:To establish a stable autoimmune rat model of POF using ovarian antigens.Methods:Healthy female rats were randomly divided into control group（n=30）and premature aging model group（n=110）.The ovarian antigen plus Freund’s adjuvant was used for subcutaneous multiple injections for a total of 3 consecutive immunizations to establish a POF model.The success rate and stability of modeling were determined by recording the regularity of the estrous cycle,observing the morphological changes of the ovaries,counting the changes in the number of follicles,and measuring the differences in serum estrogen levels.Results:Compared with the control group,rats in the POF model group showed disordered estrous cycle,atrophy of ovarian structure,increased follicular atresia,decreased number of follicles at various levels,and significantly decreased serum levels of E₂,P₄,AMH and other estrogen.Conclusion:Ovarian antigen injection can interfere with the level of estrogen in rats,damage the ovarian tissue,and build a stable autoimmune rat POF model.It provides a stable autoimmune animal model for the pathological research of POF.2.The therapeutic effect of different concentrations of h UCMSCs injected into the tail vein on POF in ratsObjective:The experiment was conducted to explore the possibility of treating autoimmune rats with POF through tail vein injection of h UCMSCs,and compare the therapeutic effects of different transplant doses.Methods:The successfully modeled POF rats were randomly divided into 4 groups（n=15）,including three different concentrations of h UCMSCs（h UCMSCs 0.25×10⁶/ml,1.00×10⁶/ml and 4.00×10⁶/ml）were injected into the tail vein with saline as the vehicle control.Compared with the ovarian shape,numbers of follicles,estrous cycles and estrogen levels between different groups,and detect cell proliferation and apoptosis in ovarian tissue to determine the therapeutic effect.Results:The h UUCMSCs system was established.After treatment,the estrous cycling of POF rats became normal,the morphology of the ovarian tissue was obviously improved,the estrogen level gradually returned to normal,and the cell proliferation and apoptosis of the rat ovary were significantly reduced.Conclusion:h UCMSCs can have a therapeutic effect on POF rats,and different doses of h UCMSCs have different improving effects on POF rats.As the injection dose increases,the ovarian function of POF rats improves significantly.3.The therapeutic effect of h UCMSCs in situ ovarian injection on POF in ratsObjective:To explore the possibility of treating autoimmune rat POF by in situ injection of h UCMSCs into the ovary,and compare the effect of different transplantation methods.Methods:The successfully modeled rats were divided into the in situ injection group（n=9）（h UCMSCs4×10⁵/25μL）and the POF control group（n=6）,and the rats without any treatment were used as the blank control group（n=15）.The therapeutic effect can be judged by observing ovarian morphology,counting the regular amount of follicles and number of estrous cycles,estrogen levels,and cell proliferation and apoptosis in the ovaries.Results:After ovarian orthotopic h UCMSCs treatment of POF rats,the ovarian atrophy of rats improved,the number of follicles at various levels increased,the serum estrogen levels increased,the granulosa cells in the ovarian tissue increased significantly,and the apoptosis decreased.The treatment effect was not much different from the tail vein injection method.Although it will cause trauma,it increases the local injection concentration and saves reagents.Conclusion:The ovarian in situ injection method further verified the improvement effect of h UCMSCs on the POF of rats.It provides a reference for the selection of different transplantation methods.4.Preliminary exploration of the mechanism of h UCMSCs on rat POF.Objective:To verify whether h UCMSCs can affect the ovaries through blood circulation,and to explore the potential mechanism for improving POF.Methods:Healthy female SD rats were injected with high-dose green fluorescent protein（GFP）-labeled h UCMSCs into the tail vein,and the PBS injection group was used as a blank control group.Samples were taken at 24,48 and 72 h after injection,and frozen sections of the ovaries were made to observe the fluorescence.By detecting the serum biochemical indicators of POF rats treated with h UCMSCs and the expression of genes affecting follicular development in their ovaries,the therapeutic mechanism of POF of h UCMSCs was further explored.Results:Green fluorescence was not detected in the control group,and green fluorescence was detected in the tracer group.The serum triglyceride（TG）and high-density lipoprotein（HDL-C）levels of POF rats treated with orthotopic transplantation of h UCMSCs increased significantly.h UCMSCs transplantation down-regulates Caspase-3m RNA and up-regulates the gene expression of Bcl-2,AMH and FSHr.Conclusion:h UCMSCs can reach the ovary through blood circulation to play a therapeutic role,promote gene expression that is conducive to follicular development,thereby increasing ovarian reserve and reducing apoptosis.It laid the foundation for a deeper exploration of the mechanism of h UCMSCs in the treatment of POF.In summery,these results further validated the potential mechanisms of promoting the release of cell growth factors and enhancing tissue regeneration,and provide a theoretical basis for the clinical application of stem cells in the treatment of premature ovarian failure.