| Background:The varying degrees of immune dysfunction were induced by the treatment of chemotherapy, radiation therapy, especially allogeneic hematopoietic stem cell transplantation (HSCT) on hematopoietic malignant diseases. In addition, by virtue of extensive application of nuclear technology, the occurrence possibility of radiation disease would be increased gradually. Patients of acute radiation sickness (ARS) are inclined to severe infection induced death due to marrow failure, multiple organ damage and immune dysfunction. In spite of hematopoietic function of radiated patients move to better situation by the therapy of hematopoietic stem cell transplantation and cytokine, high dosage of using cyclophosphamide, total body irradiation (TBI), anti-thymocyte globulin (ATG), as well as ciclosporin for graft-versus-host disease (GVHD) after post-transplant treatment and other immunosuppressive agents, will result in severe immune deficiency and secondary severe infection in transplant conditioning phase. Lymphoid progenitor cells generated from bone marrow moving into the thymus, which regarding as central immune organ. Lymphoid progenitor cells plays an important role in maintaining and regulating body immune system, after its differentiation, well development to mature T cells, migrating out of the thymus and located in the peripheral lymphoid tissues. However, as one of highly sensitive organs to pretreatment of hematopoietic stem cell transplantation and radiation, the thymus is always involved in injuring of body’s immune function after the radiotherapy, chemotherapy, transplant or severe ARS. Therefore, rapidly recovery of damaged thymic function will effectively improve the immune function of the patient. Mesenchymal stem cells (MSCs) have effects of directed differentiation, hematopoietic support and immunomodulatory. The MSCs has negative regulatory function to T lymphocyte mediated immune response in many of in vitro and in vivo studies. Meanwhile, other reports demonstrate that MSCs has a positive regulatory role on T lymphocyte differentiation, maturation, possessing a significant role in maintaining the thymic microenvironment, promoting maturation of T lymphocytes and the recovery of cell number in thymus and spleen of irradiated mouse. Whether MSCs can play a repairing role in damaged thymic function? What is the essential mechanism? Whether it can be regarding as a novel treatment to promote immune reconstitution? These are most questions uncovered yet, which will be discussed and explored in this thesis. T cell-mediated immunity participating in many processes of disease, MSCs has been used to relieve GVHD and induce immune tolerance in hematopoietic stem cell transplantation. In order to discovering the MSCs effect to immune and hematopoietic system, researches have been uncovered MSCs in clinical applications on T-cell lymphoma, myelodysplastic syndrome and etc, which will be useful clinical studies for the next preliminary discussion.Object:To establish a thymus injury model of radioactive mouse, to study MSCs repairing effect and mechanism in thymus damage, discovering novel treatment to immune reconstitution after chemotherapy and hematopoietic stem cell transplantation, offering preliminary clinical discussion for the effect of MSCs on immune and hematopoietic system in hematologic diseases.Methods:1. Based on percoll separation and adherence screening method, we modified previous MSCs culture ways, successfully separated and purified bone marrow MSCs from male C57mouse. To verify whether the cultured cells are the bone marrow MSCs according to the cell morphology, cell phenotype and directed differentiation experiments. 2. Selecting female BALB/C mice with Co6O y-ray irradiated in6Gy,9Gy,12Gy three dosages as thymus radiated injury model. Respectively irradiating mice mediastinal site with three dosages from the chest with remaining parts covered by10cm thick lead shelter. Dosage is154.7cGy/min, mice are80cm away from the radiation source, and the irradiation time is257seconds,386seconds, and514seconds separately. Body weight, thymus index, thymus tissue pathology, peripheral blood and peripheral blood T lymphocyte subsets changes are observed after irradiation. Selecting the best radiation dosage based on above results, building up a thymic injury model, which will be regards as study sample for thymic function recovery for MSCs treatment.3. MSCs are injected to thymus damaged mouse model via the tail vein, the observed index including the changes of thymus index, peripheral blood, thymus T lymphocyte subsets, peripheral blood T lymphocyte subsets, the distribution of CM-DiI labeled MSCs in the thymus after infusion of irradiated mice, the gene expression of Sex-determining Region of Y-chromosome, the P53protein expression in thymus from immunohistochemical detection are investigated. Studies on MSCs colonization in damaged thymus, the repairing effect and mechanism of MSCs treatment on thymus function.Results:1. Isolated, cultured mouse bone marrow MSCs start to adherent after24hours planting. At the beginning, MSCs appears in small round or polygonal morphology, while spindle morphology shown after cultured for7days. MSCs tend to unity, long spindle morphology when passages to the second generation. High expression of CD44, CD105and low expression of CD34, CD45in the second generation of MSCs are investigated by flow cytometry. In the directly induced environment, MSCs can differentiate into osteoblast and into fat.2.Compared with the control group, the mice body weights are decreased after irradiation in three dosages at different time points. The6Gy irradiated group has statistically significant difference (P<0.05) comparing to the control group only after14days irradiation. The9Gy irradiated group has statistically significant difference (P <0.05) comparing to the control group after14and21days irradiation. The12Gy irradiated group has statistically significant difference (P<0.05) comparing to the control group after14,21and28days irradiation.3.The thymus index of three irradiation groups after irradiation in each different time point were lower than the control group;The9Gy irradiated group has statistically significant difference (P<0.05) comparing to the control group after7,14,21and28days irradiation. The6Gy irradiated group has statistically significant difference (P <0.05) comparing to the control group after14,21and28days irradiation. Pathological examination showed mice thymus atrophy in irradiation group degeneration and of lymphocytes in cortex, thymus corpuscles disappearing in medulla, and more significant seven days after irradiation. the degree of damage are positively related;with the radiation dose. Thymic volume increases, the number of thymocytes began to gradually increase fourteen days after irradiation. Part of mice from12Gy irradiation group died28days later. Peripheral white blood cells and lymphocytest of three irradiated group were lower than the control group after irradiation in all time points (P<0.01).After irradiation, thymic CD4+CD8+cells decreased the most obviously.CD4-CD8-cells increased after irradiation. CD4+CD8-and CD4-CD8+cell ratio decreased after irradiation.Around T cell subpopulation in the peripheral blood, the percentage of CD3+CD4+cells, CD3+CD8+cells and CD4/CD8ratio continued to decline. And the same trend appeared in thymus T cell subpopulation.The injury of thymus gland can be obviously reflected in9Gy irradiation group without serious complications after irradiation, so9Gy irradiation group was selected to act as models of radiation injury of thymus.MSCs infusion help to repair thymus irridiation injury of mouse. The thymus index decreased both in the treatment group and in the irradiation control group after irradiation. But the treatment group showed less decrease.Peripheral white blood cells and lymphocytest decreased both in the treatment group and in the irradiation control group after irradiation.Less decrease from the treatment group.A fter irridiation,CD4+CD8-cells, CD4-CD8+cells and CD4+CD8+cells decreased less than the the irradiated control group. Changes of T cell subsets in peripheral blood was similar.CM-Dil labeled MSCs can be found in the thymus colonization in30days after infusion. In addition, Sry of the male donor mice can still be detected in thymic tissue of female recipient mice in28days after infusion. the expression of P53protein in the thymusof the treatment group was significantly lower than the control group.4.MSCs can do good to T cell lymphoma and myelodysplastic syndrome.Conclusion:1by Percoll separation and adherence screening method, also with improved the culture conditions,MSCs were successfully cultured.MSCs were identified from morphology, cell phenotype and multilineage differentiation capacity.2Dose of6Gy,9Gy,12Gy all can cause radiation damage in mouse thymus, but9Gy irradiation caused typical of thymus with no other serious complications.It was the selected dose to build models of radiation thymus injury of mice.3MSCs can home to the thymus of radiation injury mice and repair the thymus function, reduce the thymocyte apoptosis,promote the thymus immune reconstruction.MSCs can be used to reconstruct immunodeficiency.The application of MSCs in the clinical treatment of related hematologic diseases was safe and reliable.MSCs has certain clinical value for the treatment of T lymphoma and myelodysplastic syndrome. |
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