The Effect Of Intrathymic Injection With Xenogeneic Soluble Antigen In Human Ovarian Tissue Xenograft In Rabbit Model

Background:In the last few years, the number of female patients suffering from cancer is increasing, and the age of onset is younger. Continuous improvement of treatment also makes more and more young women to ask for fertility preservation. At present, cryopreservation of human ovarian tissue has become a hot spot for the preservation of female reproductive function. In the domestic and foreign studies, the effect of the transplantation of ovarian tissue in nude mice is worthy of recognition, but the relationship between the human and the nude mice is far away, the cost of feeding is higher, and the space of the graft is limited. The use of rabbits as transplantation receptors can enrich the existing transplantation model, and provide more choices for the female patients to protect reproductive functions. If we choose the rabbits as a recipient, it is necessary to overcome immune rejection to achieve long-term preservation of ovarian tissue. Since 1980s, many domestic and foreign scholars have carried out a large number of systematic studies on the induction of immune tolerance in the thymus. The method is first to use drugs or irradiation to clear the recipient’s T cells, and then by injecting heterologous antigen in the thymus, the newly formed T lymphocytes of the thymus were selected by the negative selection, and the immune tolerance of the different antigens was produced. This method is used to get more mature applications in rodents. But there are no related research reports on the immune tolerance induced by thymus injection in rabbits.Objective:To explore the effect of intrathymic injection with xenogeneic soluble antigen in human ovarian tissue xenograft in rabbit model.Method:The fresh ovarian tissue was cryopreserved by vitrification for at least four weeks. After thawing the tissue must be transplanted in 3 hours.10 female New Zealand white rabbits were randomly divided into the experimental group (n=5) and the control group (n=5), and the whole body irradiation with X rays (3Gy) was performed 1 week after castration. The thymus was injected with donor antigen (0.1ml) after 1 week of irradiation in the experiment group; the same amount of normal saline was injected in control group. One week after the injection, the frozen-thawed human ovarian tissue was transplanted in rabbit dorsal muscle. Blood serum samples were collected to detect the level of IFN-y and IL-10 before irradiation, one week after irradiation (before injection),one week after injection(before transplantation), one week, three week, five week and seven week after transplantation. And the ratio of CD4+and CD8+ lymphocytes in peripheral blood of rabbits were detected with fresh anticoagulant blood.After 8 weeks, the recovery rate of ovarian tissue was recorded and the morphology of ovary was observed. The morphology of tissue and the rate of fibrosis necrosis were observed by HE staining.Results:1、The method of ovarian tissue cryopreservation can be an effective way to preserve ovarian tissue.2、 After irradiation, the IL-10 level of the experimental group and the control group were significantly increased (p<0.05). The experimental group was reduced3weeks after transplantation, but the control group was reduced before transplantation. There was no significant difference in IFN-γ between the experimental group and the control group. The ratio of CD4+/CD8+ of the experimental group began to rise 3 week after transplantation, while the control group began to rise 1 week after transplantation.3、 Eight weeks after transplantation, the ovarian tissues were recovered, and the recovery rate was higher in the experimental group than in the control group. There were a large number of inflammatory cells infiltrated in the two groups. Compared with the estrogen level before transplantation, the estrogen level in the experimental group was significantly higher in the first 3 weeks after transplantation, and the control group was higher at 1 week after transplantation and no significant difference was observed after.Conclusions:The application of intrathymic inoculation with xenogeneic soluble antigen in human ovarian tissue xenograft in rabbit model may delay the immune rejection of the time. But we did not get a long-term immune tolerance in our study.