Human Cord Blood Mononuclear Cells (HCMNCs) Transplantation In The Treatment Of Radioactive Experimental Study Of Premature Ovarian Failure In Nude Mice.

Purpose and significance:Premature ovarian failure(POF)is the appearance of menopausalsymptoms in women younger than age 40. Women in POF frequently exhibit amenorrhea, hypoestrogenism (E2<25 pg/ml), and elevated serum gonadotropin levels (FSH>40 IU/L,LH>30 IU/ml), usually called early menopause. The etiological of POF is complicated. In early studies,the known causes include: genetic aberrations,autoimmune ovarian damage,environmental factors like virus infections,toxins and surgery on ovarian, cigarette smoking, and so on.Also some of the POF are unique. The cause of POF is complex,and the pathologic mechanism is unkown clearly, so there are no effecive therapies for POF patients. Hormone replacement therapy (HRT) immunosuppressive agents treatment, transplantation of ovarian living tissue and so on , but they are still not optimistic for POF patients.The multiple differentiation potential and low immunogenicity are the characteristics of human cord blood cells. In this study, we transplanted the human cord blood mononuclear cell (HCMNCs) to the annimal models of POF to investigate the feasibility and efficacy of the treatment. Our study will provide some experimental basis for finding reasonable and effective method for POF patients.PART1: Constructing POF animal modelsObjective:To discuss the feasibility of radioactive elements in constructing nude mouse model of premature ovarian failure (POF) and the optimal dose of radiation.Methods:1.80BALB / C nude mice , 7 or 8 weeks old , were randomly divided into A, B, C three groups.2.A group was the control group, B, C, D groups were irradiated by 0.3Gry, 0.5Gry, 0.7Gry cobalt 60γray respectively, E group, bilateral ovariectomy group. Daily vaginal smears of the line were observed the changes of estrous cycle in nude mice.3.After 15 and 30 days, 8 mice in each group were sacrificed to determin E2, FSH, LH levels in serum by ELISA, and observe the change in the number of ovarian follicles in ovarian tissues with H-E staining. Results:1. General observation: There were no significant changes in activities, eating, et, of the nude mice of A group, Significantly reduce in B, C, D group,more obviouse in E group.2. The nude mice of A group had no significant change in the estrous cycle, B, C, D groups had some disorder of different degrees in estrous cycle, while the E group estrous cycle had disappeared, the changes in estrous cycle of B, C, D, E groups compared with A group were statistically significant (P <0.05), however, B, C, D, E groups showed no significant difference (P> 0.05).3. 15 days after treatment, the serum of E2 in B, C, D, E group nude mice was significantly lower, while FSH, LH higer, compared with A group with statistical significance (P <0.05), howere, B, C, D, E groups showed no significant difference each other (P> 0.05). Along with the time, Such differences were more apparent.Conclusion:Co-60γ-ray irradiation can be successfully constructed annimal models of POF. And the optimal dose of irradiation is 0.5Gry.PART2: The effects of human cord blood mononuclear cell (HCMNCs) transplantation on the radiological experimental study of premature ovarian failure in nude miceObjective:To investigate the feasibility and efficacy of transplanting HCMNCs to treat the nude mouse models of POF by irradiation. Methods:1. 105 BALB / C nude mice ,7 or 8 weeks old ,were randomly divided into A, B, C three groups.2. A group were not treated, B, C groups were irradiated by the cobalt-60γ-ray in order to achieve the nude mice ovarian failure occurs, for establishing the annimal model of POF.3. Daily vaginal smears were used to observe the changes of estrous cycle in nude mice. After 30 days, 15 mice in each group were sacrificed to determin E2, FSH, LH levels in serum by ELISA, and observe the change in the number of ovarian follicles in ovarian tissues with HE staining. 4. HUCBC cell suspension were prepared by Ficoll density gradient centrifugation and labeled with BrdU.5. Making sure the annimal models of POF were successfully established, then the prepared HCMNCs cell suspension which labled by BrdU were injected into both ovaries of C group nude miouse, L-DMEM medium were injected into both ovaries of nude mouse of B group, however, A group nude mouse were served as healthy controls without any treatment.6. 4 weeks after transplantation, all mice in each group were killed to derermin E2, FSH, LH levels in serum by ELISA and observe changes in the number of ovarian follicles of the ovarian tissues with H-E staining and the survival of transplanted cells with BrdU histochemical staining.Results:1. General observation: There were no significant changes in activities, eating, et, of the nude mice of A group, Significantly reduce in B, C group. After transplantation of human cord blood mononuclear cells, the nude mice of C group generally improved compared with B group.2. After Cobalt 60γ-ray irradiation, B, C groups of nude mice had some disorder of different degrees in estrous cycle, while A group had no significant change. After transplantation of human cord blood mononuclear cells, the diorder of estrous cycle in C group was significantly improved, epithelial cells, keratinocytes and white blood cells alternating, the Periodicity was more regularly with time.3. 30 days after Co 60γirradiation, E2 levels in serum of B,C groups was significantly lower, and FSH, LH levels were significantly higher compared with control group A group, with statistical significance (P <0.05).4. Microscopic observation and statistical analysis showed that the number of the original follicles and primary follicles in ovarian tissue of B,C groups was significantly reduced (P <0.01) compared with A group.5. After the transplantation of HCMNCs for 4 weeks, E2 levels in serum of C group was significantly higher than POF group B , but was not significantly different compared with the control group A . However FSH, LH levels in serum of C group was significantly reduced compared with POF group B , but was not significantly different compared with the control group A .6. Immunohistochemical staining showed transplanted human cord blood mononuclear cells in the ovary of nude mice can be settled and survival. Conclusion:Human cord blood mononuclear cells (HCMNCs) can be successfully implanted in nude mice in vivo ovary and survival. They can also improve the ovary function.