The Experimental Study Of Repair Effect Of Human Umbilical Cord Mesenchymal Stem Cells Transplantation On Chemotherapy-induced Ovarian Damage

Background:As the advances in the treatment of chemotherapy, many patients with malignant tumors can remain alive for a long time, and some can even be completely cured. However, these patients after chemotherapy, such as alkylating agents, have appeared in varying degrees of menstrual disorders, acute or chronic failure of ovarian function and even infertility. The most common complication is premature ovarian failure. After chemotherapy have been applicatied, some complications would happen,such as sustained amenorrhea,genital atrophy, and the syndrome because of the increase of follicle stimulating hormone (FSH) and luteinizing hormone (LH), and the reduce of estradiol (E2). At the same time,the symptoms of perimenopausal syndrome would be Accompanied, for instance,the waves of heat, sweating, facial flushing, mood swings, low libido, vaginal dryness, and even infertility.Currently, the mechanism of chemotherapy-induced premature ovarian failure is not clear.Meirow D proposed that the cell apoptosis due to the chemotherapy drug was an important mechanism for the damage in ovarian structure and function. Because chemotherapeutic drugs may affect the activation of some specific signal in follicle which can activat and promot the cell apoptosis, the inhibition of this process is likely to inhibit apoptosis and protect the ovarian function. Tilly JL also discovered that the apoptosis of oocytes after chemotherapy may be mediated by ceramide 1-phosphate sphingosine. Sphingolipids ceramide as a molecular signal transduction plays a role in apoptosis. Morita Y found that the application of 1 sphingosine phosphate in the mouses which lacked of an enzyme for produce ceramide acid would inhibit the oocyte apoptosis due to chemotherapy drug.In addition, chemotherapy drugs may cause the fibrosis of ovarian tissue, autoimmune and other damages.In recent years, many methods have been adopted to prevent the premature ovarian failure due to chemotherapy drug. For example,Blumenfeld Z proposed that the combined treatment of GnRHa and chemotherapy drugs can significantly reduced the rate of POF in the patients with Hodgkin's disease, breast cancer or lupus kidney disease.The cryopreservation techniques were proposed, including the frozen embryos, frozen ovarian tissue transplantation and frozen oocytes and in vitro maturation, however,these techniques were unsatisfactory. With the gradual deepening researchs in stem cell, we found that the transplantation of rat bone marrow mesenchymal stem cells to treat the premature ovarian failure induced by chemotherapy in the rats could repair the ovarian structure, improved the ovarian endocrine function,such as increase of rat ovarian follicles and estradiol levels and decrease of FSH levels. Therefore, these therapeutic effects prompted that the transplantation of bone marrow mesenchymal stem cells maybe turn into a new treatment for the premature ovarian failure. Furthermore,the mechanism of these therapeutic effects maybe considered that the MSCs can secrete VEGF, IGF-1, HGF and other factors,which can inhibit the apoptosis of ovarian granulosa cell. However, there is a high degree of virus contamination to separate the bone marrow mesenchymal stem cells, the stem cell concentration is low, only a limited volume of bone marrow can be extracted, and there is an associated risk of harm to the patient during harvesting. Therefore, the overall success of BMMSCs has been limited. Therefore, people try to isolate the MSCs from fetal appendages,such as umbilical cord and placenta, which can be extensively used in clinical studies because of their wide availability, the absence of ethical concerns, and low oncogenicity and bacterial/viral contamination.In the experiment, we selected the placenta and umbilical cord from the same healthy woman who delivered a healthy full-term infant by cesarean section, for this reason, the influences of individual differences were ruled out,therefor,we can select the appropriate mesenchymal stem cells. After the establishment of rat model with premature ovarian failure due to chemotherapy in vitro, mesenchymal stem cells cells labeling with PKH26 were transplantatied by intravenous injection and local injection. The locationes of mesenchymal stem cells in ovarian after transplantation were detected by fluorescence microscopy. Regular testes of estrogen and FSH and vaginal smear test changes in rats were observed, in addition,the rats were sacrificed in groups in order to observe the pathological changes including ovarian tissue morphology and follicle count,the pathology of liver,kidney and uterus. Meanwhile,the fertility of rats and the health of future generations were observed.Chapter 1 The comparison of biological characteristics and ultrastructure between human umbilical cord mesenchymal stem cells and human placenta derived mesenchymal stem cellsObjective:To establish the methods of separation,culture of human umbilical cord mesenchymal stem cells (HUMSCs) and human placenta derived mesenchymal stem cells (PDMSCs) in vitro,and explore the phenotype of the two kinds of cells.Meanwhile,we compared the biological characteristics and the ultrastruture of human umbilical cord mesenchymal stem cells and human placenta derived mesenchymal stem cells, expecting to find out the distinctiones of MSCs derived from different sources.Methods:1 The fresh human umbilical cord and placenta were obtained from the same woman who delivered a healthy full-term infant by cesarean section.Tissue cultures were maintained in DMEM medium supplemented with 10% FBS, and were incubated in a humidified atmosphere with 5% CO2 at 37℃. Cells were subcultured every three days at a splitting ratio of 1:3.2 The rate of cell proliferation, levels of apoptosis, and secretion of various growth factors (i.e., VEGF, IGF-Ⅰ, and HGF) of the two types of cells were analysed.3 The surface morphologies of cells were studied by tapping-mode atomic force microscopy, and the cell ultrastructures were observed with a Philip CM-10 TEM.Results:1 The cells isolated from human umbilical cord and placenta exhibited an elongated spindle (fibroblast-like) morphology, abundant cytoplasm, and large nuclei, The cells readily attached to walls of the culture dishes, grew in parallel or vortex-like patterns, and then proliferated rapidly to confluence. The cells were stably passaged. Flow cytometry revealed that the cells isolated from human umbilical cord and placenta expressed CD29, CD44, CD73, CD90, and CD105, but not CD14, CD34, CD45, CD 106, CD 133, or HLA-DR (MHC-Ⅱ). These findings indicate that the cells we isolated only expressed mesenchymal-specific antigens; they did not express hematopoietic- or endothelial-specific antigens, thus confirming that the isolated cells were human MSCs.,these results mean that the cells we isolated were HUCMSCs and PDMSCs.2 The rates of the second-passage HUMSCs and PDMSCs proliferation were analyzed using the cell-counting kit-8 (cck-8) in media containing 10% fetal bovine serum but no growth factors. The rate of cell proliferation was slow during the first 2 days (latent phase), accelerated during days 3-6 (logarithmic phase), and slowed down thereafter (stationary phase). The duplication time of the cells of the second-passage HUMSCs in logarithmic phase was 2.560±0.117 and the second-passage HUMSCs was 2.956±0.204, apparently, significant difference was found in the results(P<0.05).The proliferative ability of HUCMSCs was more powerful than PDMSCs:the duplication time of the cells was shorter, the rate of apoptosis was lower, the quantity of the cell in the mitotic phase was higher,and autophagic vacuoles were more common. However,the ability of secretion of PDMSCs were more notable than HUMSCs, such as VEGF, IGF-1, HGF.3 TEM revealed that tubular cristae in the mitochondria of PDMSCs, which is similar to the ultrastructure of mitochondria in endocrine cells, suggesting the structural basis for their secretion of bioactive factors. This effect might be related to the ability of secretion of placenta to maintain the pregnancy.AFM revealed that PDMSCs had a higher quantity of large cuboidal or flat cells than HUMSCs,and the parallel myofilaments and pseudopods of PDMSCs were more intensive than HUMSCs,suggesting that PDMSCs can facilitate their adherence. At the same time,TEM demonstrated that the scattered microvilli-like structures were abundant on the surface of PDMSCs. This characteristics maybe come from the adherent function of placenta.Conclusion:The cells isolated from human umbilical cord and placenta readily attached to walls of the culture dishes, grew in parallel or vortex-like patterns, and then proliferated rapidly to confluence.Moreover,the cells expressed CD29, CD44, CD73, CD90, and CD105, but not CD14, CD34, CD45, CD 106, CD 133, or HLA-DR (MHC-Ⅱ). These findings indicate that the cells we isolated only expressed mesenchymal-specific antigens; they did not express hematopoietic- or endothelial-specific antigens.The proliferative ability of HUCMSCs was more powerful than PDMSCs.In conclusion, HUMSCs had higher self-renewal capacity than PDMSCs.However,the ability of secretion of PDMSCs were more notable than HUMSCs, such as VEGF, IGF-1, HGF.Chapter 2 The experimental study of PKH-26 labeled human umbilical mesenchymal stromal cellsObjective:To determine whether PKH26-labeling affect the morphologies, phenotypes, proliferation and secretion abilities of human umbilical mesenchymal stromal cells (HUMSCs) were investigated.Methods:Isolated HUMSCs were labeled with PKH26, and cell morphology was observed under microscope. Cell cycle, apoptotic cell death, expression of PKH26 and the proliferation rate were evaluated. Additionally, fluorescence intensity of PKH26 labeling at different passage time was quantified. Moreover, the effects of PKH26-labeling on selective growth factors:including vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) secretion were investigated.Results:There were no detectable differences in cell morphology, cell growth and proliferation rate after PKH26 labeling. The PKH26 labeling disappeared after passage six times. PKH26-labeling does not affect the morphology, proliferation, and apoptosis of HUMSCs, whereas may influence cellular growth factor secretion. It is necessary to perform further comparison study in vitro and in vivo to investigate the effect of PKH-labeling on the function of cells.Conclusion:PKH26 labeling technology may be useful for studying homing, differentiation, and proliferation of human umbilical cord mesenchymal stem cells after transplantation into rats of chemotherapy-induced premature.Chapter 3 The preliminary immunological study and comparison of two route of transplantations of human umbilical cord mesenchymal stem cells between local injection in ovary and intravenous injection in rats.Objective:In order to evaluate the effects on function of various organs in rats as a result of transplantation of human umbilical cord mesenchymal stem cells, and to understand the distribution of human umbilical cord mesenchymal stem cells in rats due to different route of administration, such as local transplantion and intravenous injection. Therefore, we could analyze the possibility of immune rejection of the transplantation of human umbilical cord mesenchymal stem cells in rats.Methods:The rats for study were divided into 3 groups,5 rats in each group, were female wista inbred rats,weighing 180-200g, vaginal smears are in normal estrous cycle. The first group was the control group without any treatment, the second group was the group of ovarian injection, the third group was the group of intravenous. injection. After the successful transplantationsof PKH26 labeled human umbilical cord mesenchymal stem cells, the distribution of human umbilical cord mesenchymal stem cells in rats was detected by fluorescence microscope. and the health conditions of the rats were also observed.Results:The results showed that both local injection in ovary and intravenous injection had no obvious acute and chronic immune rejection,such as difficulty in breathing, bleeding, hair loss, hematuria, bloody stools and other symptoms. In addition,the pathology of brain, liver, kidney, ovary and uterus had no obvious lymphocyte infiltration and proliferation of fibrous tissue, which may suggest that the transplantation of human umbilical cord mesenchymal stem cells may not cause significant immune rejection even in different animal categories. Therefore, the natural immune tolerance may exist between human umbilical cord Mesenchymal stem cells and rat organs.The phenomenon of cells aggregation was detected in the ovaries of the second grouop, except for a small amount of human umbilical cord mesenchymal stem cells was detected in the position of uterus near ovary due to the vascular communication between uterus and ovary.there was no appearance of umbilical cord mesenchymal stem cells in other organs.In addition, umbilical cord mesenchymal stem cells was detected in the ovary, uterus, liver, kidney cortex in the third group, except for the brain tissue.Conclusion:The transplantation of human umbilical cord mesenchymal stem cells in rats could not cause significant immune rejection even in xenogeneic transplantation and did not affect the experimental results, therefore,the experiments indicated that human umbilical cord mesenchymal stem cells maybe exist the natural immune tolerance in rat organs.Chapter 4 The experiment of repair effect of human umbilical cord mesenchymal stem cells transplantation on chemotherapy-induced ovarian damage in vivoObjective:To evaluate the feasibility of treatment of the human umbilical cord mesenchymal stem cells on rats with premature ovarian failure due to chemotherapy, and to assess the difference between intravenous injection and local injection.Methods:1 To Establish the rat model with premature ovarian failure due to chemotherapy in vitro.2 Human umbilical mesenchymal stem cells cells labeling with PKH26 were transplantatied by intravenous injection and local injection.3 To observe ovarian situation after transplantation of human umbilical cord mesenchymal stem cells.①regular testes of E2 and FSH and vaginal smear test changes in rats were observed;②the rats were sacrificed in groups in order to observe the pathological changes including ovarian tissue morphology and follicle count;③the locationes of human umbilical mesenchymal stem cells in ovarian after transplantation were detected by fluorescence microscopy;④the fertility of rats and the health of future generations were observed.Results:The recovery of the estrogen in the groups of ovarian injection and intravenous injection was faster than model group, but no significant difference existed between the two treatment groups. Meanwhile, the ovarian injection group was the first group to be detect the recovery of the follicle stimulating hormonerecovered the first, the intravenous injection group was the second group, model group was the last group,but there are no significant difference between the ovarian injection group and the intravenous injection group at 90 days.These results suggested that the transplantation of human umbilical cord mesenchymal stem cells could partially repaired the chemotherapy-induced ovarian damage, and the group of ovarian injection was the first group to take effect,but no significant difference existed between the two treatments group from the long-term efficacy.The results of ovarian pathology showed the transplantation of human umbilical cord mesenchymal stem cells could reduce the damage to the follicle due to the cyclophosphamide, and the group of ovarian injection was the first group to take effect,but no significant difference existed between the two treatment groups from the long-term efficacy.Meanwhile,we have observed that some rats in the two treatment groups restored reproductive function, although the number of offspring were less than the normal group, however, the results suggested that the transplantation of human umbilical cord mesenchymal stem cells could recover the ovarian endocrine function and improving the ovulation of ovarian function. In addition, the rats in the group of ovarian injection gave birth to offspring earlier than the group of intravenous injection,which related to the recovery of FSH earlier in the group of ovarian injection than the group of intravenous injection.ovarian.In addition, we observed the growth and development of young rats,however, there are no ignificant difference in the young rats between the normal group and treatment groups,all the young rats had the normal fertility, indicating that the transplantation of human umbilical cord mesenchymal stem cells does not affect the growth and development of offspring. Conclusion:The transplantation of human umbilical cord mesenchymal stem cells have a certain therapeutic value in the treatment of rats with premature ovarian failure due to chemotherapy.In this study,we found many therapeutic effects,such as the improvement in hormone levels, recovery in ovarian pathological structure in some rats and the offspring in good conditions. In addition, the outcomes of our researches prompted that there were no difference in the therapeutic effect between intravenous injection and local injection, indicating intravenous injection may become more simple, minimally invasive transplantation method in the treatment.