| Introduction: Hematopoietic inductive microenvironment(HIM) is the site of origin, proliferation, differentiation and development of hematopoietic stem cells (HSCs). As one of important component of HIM, stromal cells are involved not only in the self-renew, proliferation, differentiation and homing of HSCs and the occurrence, progress and prognosis of hematological diseases, but also in immunological regulation. So, it is very important to study the effect on hematoposis and immunological regulation of stromal cells.Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is an effectively curative therapy for a variety of hematologic malignancies. Genotypically HLA-identical sibling donors are only available for about 30% of patients. The HLA-haploidentical hematopoietic stem cells transplantation could made patients gain the chance of transplantation for about 90% patients. However, the use of mismatched allograft has been disappointing due to the high incidence of graft-versus-host disease (GVHD), delayed immunogical and hematopoietic reconstitution and infectious complications resulting in an unacceptable transplant-related mortality (TRM) and mortality. It is pivotal for clinic practice to decrease TRM and improve survival rate for patients of HLA-haploidentical hematopoietic stem cells transplantation through the regulation of GVHD.Our prevous study showed that the hematopoietic stromal precursor cells exist in umbilical cord blood, and human umbilical cord derived-blood stromal cells (hUCBDSCs) could be effectively expanded by definite cytokine combination in vitro and the cultured hUCBDSCs could expand HSCs. However, we found that the culture of hUCBDSCs is extremely difficulty. It is a new direction to explore the role of immunogical regulation for hUCBDSCs. This study firstly explored the optimal conditions for the culture of hUCBDSCs, then the immunogical regulation of hUCBDSCs on mice in vitro was detected, and further study on the hematopoieic reconstruction and the regulation of GVHD in mouse HLA-haploidentical hematopoietic stem cells transplantation in vivo was carried out.Methods:1. The two step separation procedure of 6% gelatin followed by Percoll and 3% gelatin followed by Ficoll was used to separate the mononuclear cells from human umbilical cord blood, and modified Dexter culture system was used to cultured hUCBDSCs. The growth of hUCBDSCs in different sepataion methods was observed with invert microscope and the morph was examined by wright's stain, Immunocytochemistry and cytochemistry were used to evaluate the cell components of hUCBDSCs. The proliferation was assayed by CCK-8.2. The proliferation of hUCBSCs for spleen lymphocytes of mice in vitro was detected by CCK-8 in mixed culture. The cell cycle was examined by flow cytometry (FCM). The FCM and fluorescence immunocytochemistry were used to detect the expression of CD49b NK cells,CD3 T cells and adhesion molecule CD49d. IL-4 and IFN-γin the supernatant of MLR were assayed with enzyme linked immunosorbent assay (ELSIA). The adhesion of lymphocytes to hUCBDSCs in mixed culture was further study by scanning electron microscope and fluorescence immunocytochemistry. From this study we explored the immunological regulation of hUCBDSCs in vitro and the human bone stromal cells (hBMSCs) were used as control.3. The GVHD model of mouse HLA-haploidentical hematopoietic stem cells transplantation was constructed by parental generation (C57BL/6 (H-2d♀)) into F1 (H-2b×d ) filial generation (BABL/C (H-2b♂)×C57BL/6 (H-2d♀)). The per F1 recipient received 1×107 bone marrow cells and 1×107 spleen lymphocytes from the donors after receiving 60COγ-ray total body irradiation. The survival time, chimeric rate, clinical manifestation and pathological change of GVHD were observed.4. 1×106 hUCBDSCs was infused to the recipient of mouse HLA-haploidentical hematopoietic stem cells transplantation. The hematopoietic reconstruction was explored and the ablities of CFU-GM, CFU-E and CFU-Mg in bone marrow of transplantation mice were observed.5. 1×106 hUCBDSCs was infused to the recipient on the basis of GVHD model. The GVHD was observed. The expression of CD49b NK cells,CD3 T cells and CD49d in spleen of recipients mice was examined by FCM, and IL-4 and IFN-γwere assayed by ELSIA in the serum of peripheral blood in different time posttransplantation. Fluorescence immunohistochemistry was used to detect the expression of adhesion molecule CD49d in the target tissues of GVHD.6. The migration and distribution of hUCBDSCs marked with CM-DiI was inspected in mouse HLA-haploidentical hematopoietic stem cells transplantation.Results:1. The optimal condition for the culture of hUCBDSCs: 6% gelatin was much better in the sedimentation of red blood in human umbilical cord blood than 3% gelatin. The recovery rate of MNCs and CD34+ cells in 6% gelatin followed by Percoll was higher than that in 3% gelatin followed by Ficoll (p<0.05). The proliferation, the numbers of adhension cells at 48 hours, the time of initial expansion, the initial form of stromal cells colonies and the maximum time of strmal cells, the time of primary culture and the culture successful rate separated with 6% gelatin followed by Percoll were better than that 3% gelatin followed by Ficoll. There were no different in cytochemistry, immunocytochemistry and the morph of cells between methods.2. The immunological regulation of hUCBDSCs in vitro: hUCBDSCs promoted the proliferation of lymphocytes came from mice spleen and the role is much stronger when hUCBDSCs combined with recombinant human IL-2(p<0.05), however, the role of proliferation was not detected by the culture supernatants of hUCBDSCs. hUCBDSCs significantly increased the ratio of S phase and G2/M phase (p<0.05). hUCBDSCs and hBMSCs increased the expression of CD49b NK cells, and decreased the expression of CD3 T cells and adhesive molecule of CD49d (VLA-4), however, hUCBDSCs was much stronger than hBMSCs (p<0.05). It is much stronger in promoting the expression of IL-4 and decreasing the expression of IFN-γin hUCBDSCs than that in hBMSCs (p<0.05). It was detected that both hUCBDSCs and hBMSCs could inhibite the expression of CD49bIFN-γNK1 cells and CD49bIL-4 NK2 cells. The adhesive cells mainly consisted of large lymhocytes became few with the delay of time and the lymphocytes had adhered with the hUCBDSCs layer by parapodium and some of them had niched the mesh consisted by the confluence of hUCBDSCs, even migrated to underlayer of hUCBDSCs. The CD3 T cells and CD49b NK cells was fewer in seventh days after mixed culture than in third days.3. The GVHD model of mouse HLA-haploidentical hematopoietic stem cells transplantation was successfully constructed.4. hUCBDSCs promote the hematopoietic reconstruction of mouse HLA-haploidentical hematopoietic stem cells transplantation: hUCBDSCs accelerated the hematopoietic reconstruction of mouse HLA-haploidentical hematopoietic stem cells transplantation (p<0.05).5. The immunological regulation of hUCBDSCs in GVHD of mouse HLA-haploidentical hematopoietic stem cells transplantation: hUCBDSCs significantly decreased the mortality and prolonged the survival time of mice (p<0.05). The degree of GVHD was much lower in hUCBDSCs group than that without hUCBDSCs (p<0.05). hUCBDSCs group significantly promoted the expression of IL-4 and inhibited IFN-γin the serum of blood (p<0.05). The hUCBDSCs group significantly increased the expression of CD49b NK cells, and decreased the expression of CD3 T cells and adhesive molecule CD49d (p<0.05). A significant reduction of VLA-4 labeling in the target organs of GVHD was evident in mice infused with hUCBDSCs (p<0.05).6. hUCBDSCs marked with CM-DiI shifted to all tissues/organs in the early phase in mouse HLA-haploidentical hematopoietic stem cells transplantation, the continued expression was detected in bone marrow, immunological organ spleen and target organ of GVHD and the expession was little in other organs.Conclusion1. Modified 6% gelatin followed by Percoll was the optical separation procedure for the culure of hUCBDSCs.2. hUCBDSCs accelerated the hematopoietic reconstruction of mouse HLA-haploidentical hematopoietic stem cells transplantation.3. hUCBDSCs mainly shifted to bone marrow, spleen and target organs of GVHD.4. hUCBDSCs alleviated the degree of GVHD in mouse HLA-haploidentical hematopoietic stem cells transplantation by stimulating NK cells and inhibiting T cells proliferation, increasing the expression of IL-4 and decreasing the expression of IFN-γand adhensive molecule of CD9d (VLA-4). |
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