| Experimental study on CD34+ cell mixed with TK+T lymphocyte transduced TK gene transplantation for treatment of graft-vurses-host disease and preservation of graft-vurses-leukemia effects in H~2 haploidentical murine modelsA great deal of achievements have been attained in allogeneic hematopoietic stem cell transplantation. However, there are a plenty of patients who could not be transplanted because of lack of HLA-identical donors. HLA-haploidentical transplantation makes many patients with hematological malignancies get transplanting opportunities. Application of HLA haploidentical transplantation is limited because of high morbidity and mortality related to transplantation, such as GVHD, fatal infections and so on. With the technologic development of HLA haploidentical CD34 + cell transplantation, morbidity and mortality of GVHD dramatically decrease and more donors are available. Early transplant-related mortality falls due to reduced immunosuppressive agents and alleviated pulmonary and hepatic toxicity. However, T-cell depletion leads to weakened GVL effects and increased incidence of leukemic relapse. The fatal infections occur because of patient' s declined cellular immunologic function post- transplantation. The overall survival of patients don't improve compared to non-T-cell depletion transplantation. Thus, how to preserve therapeutic effects of T lymphocytes and eliminate side effects is a urgentquestion to be resolved in HLA haploidentical CD34 + cell transplantation.Donor T lymphocytes transduced by TK gene(suicidal gene) become TK + T lymphocytes, and then CD34+ cells mixed with such TK+ T lymphocytes are transplanted to the recipients. TK+T lymphocytes play the role of immunological reconstitution, GVL effects and prevention of infections. If GVHD occurred, GCV would be administered in order to kill TK+T lymphocytes selectedly and to control GVHD, while preserving therapeutic effects of T lymphocytes. So, the advantages of transplantation of HLA haploidentical CD34+ cells mixed with TK + T lymphocytes can be preserved and the disadvantages will be overcome. Based on the assumption, we researched the transplantion of H-2 haploidentical CD34+ cells mixed with TK+T lymphocytes in H-2 haploidentical murine models, in order to provided data for clinical application of transplantion of HLA haploidentical CD34+ cells mixed with TK+T lymphocytes in treatment of hematological malignancies. ObjectiveTo establish GVHD models of transplantation of H-2 haploidentical CD34+ cells mixed with TK+T lymphocytes and models of GCV treating GVHD , and to observe GVL effect of the transplantation. In order to provide experimental basis for clinical application of transplantation of HLA haploidentical CD34+ cells mixed with TK+T lymphocytes. Methods1. Enhancement of retroviral vector titer GP+E86 packaging cells were treated by tunicamycin at 120ng/ml for 18 hours and transduced by retroviral supernatants collected in advance. Retroviral supernatants were collected to test the titer.2. Selection of transducing methods Efficiencies of four kinds of transducting methods which included coculture, centrifugating, virosomes and standard transduction were tested , in order to select one kind of method which was characterized with higher-efficiency and easy operation.3. Establishment of GVHD model of transplantation of H-2 haploidentical CD34+ cells mixed with TK+T lymphocytes Donors were C57BL/6 mice (H-2d ), and recipients are CB6F1 (H-2d/b? mice. Recipient CB6F1 mice received lethally irradiation withCo(9.0Gy)4 ~ 6h prior to transplantation. Donor splenic T lymphocytes were stimulated by murine interleukin 2 (mIL-2) and ConA, transduced by TK gene, selected by G418. CD34+ cells were separated from marrow mononuclear cells by immunomagnetic beads. Then both of them were mixed to infuse into recipient mice.4. Establishment of the models of ganciclovir(GCV)to treat GVHD To compare the therapeutic effects of two kinds of usage, GCV was injected intrapertoneally at a dose...
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