| Part ?: Establishment of a viable murine umbilical cord blood stem cell transplantation (UCBT) model.Objective: To explore the isolation of murine umbilical cord blood and establishment of a viable murine umbilical cord blood stem cell transplantation (UCBT) model. Methods: Female C57BL/6 mice were treated with 5IU PMSG i.p., female C57BL/6 mice were mated with male C57BL/6 mice at a ratio of 2:1. We humanely killed E19.5d pregnant mice. We collected umbilical cord blood with 0.4% sodium citrate supplemented RPMI-1640 complete medium. Thirty-two BALB/c mice were assigned to four groups with 8 mice per group. Recipient mice were reconstituted with PBS, 1×10~6, 2×10~6 or 3×10~6 umbilical cord blood TNCs。Platelet concentrate support with 1×10~9 platelets was incorporated in parts of experiment.Results: Average number of peripheral blood TNCs is 9.37×10~5 (range, 6.4×10~5 to 11.4×10~5) per fetus by using anticoagulant-supplemented media. BALB/c mice received TBI and PBS treatment died of hematopoietic failure between + 9.5 and +14 day post TB. The 100-day survival rates of C57BL/6 mice reconstituted with 1×10~6, 2×10~6 UCB TNCs were 87.5% (7/8) and 100% (8/8), respectively.The 100-day survival rates of BALB/c mice reconstituted with 1×10~6, 2×10~6, 3×10~6 UCB TNCs were 25.0% (2/8), 37.5% (3/8) and 37.5% (3/8), respectively(χ~2=0.4990, P=0.7792. There was not a trend of prolonged survival time with increasing UCB NCs inoculation. The 100-day survival rates of BALB/c mice reconstituted with 1×10~6, 2×10~6, 3×10~6 UCB NCs in the second experiment cohort were 87.5% (7/8), 100.0% (8/8) and 100.0% (8/8), respectively. No GVHD was observed post UCBT.Conclusion: We had modified a protocol with anticoagulant supplemented medium to collect murine umbilical cord TNCs. We established a viable murine allogeneic umbilical cord blood transplantation model.PartПEstablishment of leukemia-bearing murine allogeniec umbilical cord blood hematopoietic stem cell transplantation model Objective: To establish C57BL/6→BALB/c leukemia-bearing murine allogeniec umbilical cord blood hematopoietic stem cell transplantation model.Methods: BALB/c mice were inoculated with 1×10~6 EL9611 cells 4 days prior to TBI conditioning. BALB/c mice were randomly assigned to four groups, no TBI, TBI, syngeneic UCBT control, allogeneic UCBT. Recipient mice were reconstituted with 2×10~6 UCB TNCs with platelet concentrate support on +8day post UCBT.Results: All mice died of leukemia within 14d with no TBI. All mice died of aplasia within 14d with TBI. All mice of syngeneic UCBT group died of leukemia relapse within 20d. 62.5%(5/8) of mice of allogeneic UCBT group died of leukemia relapse within 20d, 37.5%(3/8) mice survived 100d post UCBT. The survival time of four groups are significant different (χ~2=29.24, P < 0.0001). Allogeneic group had a prolonged survival than syngeneic group (χ~2= 9.545, P= 0.0020).Conclusion: We established a viable C57BL/6→BALB/c leukemia-bearing murine allogeneic umbilical cord blood hematopoietic stem cell transplantation model with platelet concentrate support.PartШThe effects of KGF on immune reconstitution and leukemia relapse post allogeneic umbilical cord blood transplantationObjective: To explore the effects KGF on immune reconstitution and leukemia relapse post allogeneic umbilical cord blood transplantation.Methods: Real-time quantitative PCR was used to assay TREC in splenocytes. RT-PCR and runoff PCR were used to assay TRBV family spectrum. FCM was used to assay splenic lymphoid immune reconstitution.In the immune reconstitution model, twenty-four BALB/c mice were randomly assigned to allogeneic UCBT PBS control group and KGF treatment group. KGF group mice received KGF treatment s.c. 3 days prior to TBI for 7 consecutive days. Mice were reconstituted with 2×10~6 UCB TNCs with platelet concentrate support on +8 days. In the leukemia model, forty-four leukemia-loaded BALB/c mice were randomly assigned to allogeneic UCBT PBS control group and KGF treatment group. KGF group mice received KGF treatment s.c. 3 days prior to TBI for 7 consecutive days. All mice were reconstituted with 2×10~6 UCB TNCs with platelet concentrate support on +8 days.Results: The total splenocytes, T cell, NK cell and B cell per mouse of mice on +35day in PBS treatment group were (3.51±0.31)×10~7, (9.32±0.48)×10~6 and (1.59±0.11)×10~6 and (18.74±2.01)×10~6 , respectively. The splenic T, NK cell counts in KGF treatment group were higher than those of PBS control group. The sjTREC level of PBS control group was 182.2±10.7 per 105 cells, while the sjTREC level of KGF treatment group were 224.2±9.6 per 105 cells, respectively. 75.0 percent of KGF treated mice (9/12) survived +100 d and 25.0 percent (3/12) died of leukemia. 33.3 percent of PBS treated mice (4/12) survived +100 d and 66.7 percent (8/12) died of leukemia. KGF group mice had prolonged survival than PBS control group (χ~2= 4.996, P= 0.0254).Conclusion: KGF pretreatment prior to TBI could protect thymus tissue, promote T lymphoid immune reconstitution and enhance graft-versus-leukemia effect in leukemia-loaded murine allogeneic UCBT. |
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