Experimental Study On Nonmyeloablative Allogeneic Bone Marrow Transplantation For Treatment Of Leukemia

PART Ⅰ EXPERIMENTAL STUDY ON NONMYELOABLATIVE ALLOGENEIC BONE MARROW TRANSPLANTATION FORTREATMENT OF LEUKEMIA OF L7212 LEUKEMIA MICEObjectiveTo establish strategies for preventing GVHD and reducing treatment-associated complications and mortalities while preserving GVL effect in nonmyeloablative allogeneic bone marrow transplantation, as well as donor lymphocyte infusions after nonmyeloablative allogeneic BMT.Methods5 ×10~6 bone marrow cells mixed 1.5 ×10~7 spleen cells from a BALB/C mice(male) were transplanted into the nonmyeloablative irradiate inbred 615 mice(female) which received a single subcutaneous injection of 1×10~6 L7212 leukemia cells two days before(called L7212),and donor spleen cells were infused after BMT. The experimentswere designed as follows(ten mice each group):①normal control group: normal 615 mice recived the regemin containing 5Gy of totalbody irradiation(TBI) on day 0 and 200mg/kg cyclophosphamide(CTX) of inoculationintraperitioneally on day +2.②The nonmyeloablative BMT blank group: L7212 mice received TBI of 5Gy andCTX on day +2.③The myeloablative BMT group: L7212 mice received TBI of 8.5Gy and CTX onday +2, 5×10~6 bone marrow cells mixed 1.5×10~7 spleen cells from a BALB/Cmice(male) were transplanted into L7212.④The nonmyeloablative BMT group: L7212 mice received TBI of 5Gy and CTX onday +2, 5×10~6 bone marrow cells mixed 1.5 ×10~7 spleen cells from a BALB/Cmice(male) were transplanted into L7212.⑤DLI group: L7212 mice received TBI of 5Gy and CTX on Day +2, 5×10~6 bonemarrow cells mixed 1.5 ×10~7 spleen cells from a BALB/C mice(male) weretransplanted into L7212, and 5×10~6, 1×10~7, 2×10~7 spleen cells were administeredintravenously on Day +7,+14,+21.GVL effects were assessed by oberserving survival, survival rate, changes of white cells and L7212 cells in peripheral blood, colony-forming unit-spleen(CFU-S), granulocyte-macrophage colony-forming unit(CFU-GM) and histological changes; GVHD was assessed by oberserving signs of weight loss, ruffled fur, diarrhea and histological changes of skin, liver and small intestines; chimeria was detected usingcytogenetic analysis and PCR methods.Results1. The survival period of each group①The mice of normal control group reestablished hematopoiesis in 15 day, the livability of this group was 100%.②The survival period of nonmyeloablative BMT blank group was (14.7±3.4) days, but all the mice died of relapsing of leukemia.③The survival period of myeloablative BMT group was (18.3±3.2) days, which was no longer than blank group(p>0.05).@The survival period of nonmyeloablative BMT group was (22.3±4.8) days, which was longer than blank guoup and myeloablative group(P<0.05), all the mice had no obvious GVHD signs and histological changes.?The survival period of DLI group was (34.3±2.5) days, which was longer than the other three groups(P<0.05), and the mice had no obvious GVHD signs and histological changes.2. Signs of GVHD of each group60% mice in myeloablative BMT group developed signs of weightloss, hunched posture, decreased activity, fur ruffling, skin lesion, etc, and had GVHD proofs of histological changes, while nonmyeloablative BMT group and DLI group had no obvious signs aboved.3. The reestablished hematopoiesis(DCFU-S: On day 11, the numbers of CFU-S of myeloablative BMT group andnonmyeloablative BMT group respectively were (8.4±1.2) and ( 9.8±0.3 ) (P>0.05) ?(2)CFU-GM: On day 7, the numbers of CFU-GM of myeloablative BMT group andnonmyeloablative BMT group were (31.5±2.4) and (33.1±0.4) (P>0.05) ,respectively o?Changes of peripheral blood cell: WBC of mice in each group decreasedto the lowest point on day +7, and WBC in nonmyeloablative group mice washigher than myeloablative group.(4)The weight of all mice in each group began to decrease after BMT, micein myeloablative and nonmyeloablative BMT groups lost weight until their death in2-3 weeks after BMT, mice in DLI group lost weight on day 6 and resumed to normallevel on day 21.4.BMT-related complications: 40% mice in myeloablative BMT group died ofBMT-related complications in two weeks, but nonmyeloablative BMT and DLIgroups remained to live.5.Detecting of chimerismThe rates of allogeneic chimeras detected by chromosome banding analysis of bonemarrow cells were 100%,80% on day 14 and 100%,0 before death. The allogeneic chimerism were exhibited by using PCR analysis of peripheral blood lymphocytes in all three groups on day 14, and mixed chimerism in DLI group were converted to complete chimerism after one week after DLI.Conclusionsl.Intensing immunosuppression, increasing hematopoietic stem cells and immunoactive cells promoted engraftment of allograft. 2.The potential GVL effects could be preserved in nonmyeloablative BMT. 3.Nonmyeloablative BMT could reduce the GVHD-related morbility and mortality. 4.DLI after nonmyeloablative BMT could reinforce GVL effects. 5.Nonmyeloablative BMT could reduce transplantation-related complications and mortalities. PART II A CASE ANALYSIS OFNONMYELOABLATIVE ALLOGENEIC HEMATOPOIETICSTEM CELL TRANSPLANTATIONObjectiveTo evaluate the short-term efficacy, the toxicity and the complications of nonmyeloablative allogeneic hematopoietic stem cell transplantation in the treatment of myelodysplastic syndrome, and to identify its role in the combined modality treatment. MethodsA relapsed myelodysplastic syndrome patient was enrolled in this study. First, the recipient achieved complete response after induction treatment. The donor was his sister, whose HLA had 4 matched locations, and had the same blood type to the recipient. The donor was mobilized by recombinant human granulocyte colony-stimulating factor and the peripheral hematopoietic stem cells were collected and infused to the recipient. The conditionging regimen included fludarabine, anti-lymphocyte globine, cyclophosphamide, mycophenolic acid and cyclosporine A. Donor lymphocyte infusions was performed in the base of the chimera in order to enhance the graft-versus-leukemia effect.ResultsThe patient reestablished bone marrow hematopoiesis one month after transplantation and had no obvious toxities and side effects. Donor lymphocyte infusions was performed 3 months after transplantation, and the bone marrow of the patient is still in complete response until now.ConclusionsThe conditioning regimen in the nonmyeloablative allogeneic hematopoietic stem cell transplantation was reduced, and the patient tolerated well and had no obvious toxities and side effects, the reestablished hematopoiesis also accelerated. Thegraft-versus-leukemia effect was enhanced by donor lymphocyte infusions, it was a new conception of hematopoietic stem cell transplantation and had a broad applicative prospect. PART III EFFECTS OF ALLOGENEICTRANSPLANTATION OF T CYTOTOXIC-2 CELLSON GRAFT-VERSUS-HOST DISEASE ANDGRAFT-VERSUS-LEUKEMIA EFFECTObjectiveTo explore wheather the graft-versus-host disease could be decreased and graft-versus-leukemia effect be retained by transplantation of allogeneic T cytotoxic-2(Tc2) cells. Methods1. Production and detection of Tc2 cells. In vitro using IL-4, ionomycin, ConA to educate the BALB/C CD8+ T cells to turn to Tc2 cells.Using ELISA to make sure that the cells produced IL-4.2. Allogeneic Tc2 transplantation: established mice model of acute lymphoid leukemia (as described above), the nonmyeloablative conditionging regemin included TBI 5Gy, CTX 200mg/kg of inoculation intraperitioneally on day +2, the experiment was designed as followed.?control group: L7212 mice reciving nonmyeloablative regemin received 0.5mlnormal saline.?chemotherapy group: L7212 mice received CTX (60mg/kg/d X 6d)intraperitioneally.?The nonnmyeloablative BMT group: L7212 mice received the nonmyeloablativeconditioning regemin, lxlO7 bone marrow cells mixed 2xl07 spleen cells from aBALB/C mice(male) were transplanted into L7212.@Tc2 BMT group: L7212 mice received the nonmyeloablative conditioning regemin,lxlO7 bone marrow cells mixed 2xl07 Tc2 cells from a BALB/C mice(male) weretransplanted into L7212.3. Detecting killing effect of Tc2 cells on L7212 leukemia cells by MTT.