OBJECTIVES: To establish mouse bone marrow transplantation by pretreatment with chemotherapy, and to explore the dynamic changes of immune cells in the early stage of allogeneic transplantation in the spleen of mice. METHODS: Mice were divided into 4 groups (80 mg/kg group, 100 mg/kg group, 120 mg/kg group, and 150 mg/kg group) according to the difference in dose of busulfan. The mice were treated with busulfan and cyclophosphamide combined chemotherapy, and the appropriate dosage was determined by evaluating the myeloablative effect and drug toxicity. According to the type of the genetic transplantation, the mice were also divided into 4 groups: An allogeneic transplantation group, a homogenic transplantation group, a chemotherapy alone group, and a normal control group. The mice were pretreated with busulfan and cyclophosphamide before bone marrow transplantation. In the allogeneic transplantation group, the suspension of splenocytes was prepared at the first day, the 3rd day, the 5th day, and the 8th day after transplantation for flow cytometry detection, and the dynamic changes of splenic immune cells were analyzed. The homogeneic transplantation group served as the concurrent control, the normal control group served as the control of basic value of spleen immune cells, and the chemotherapy alone group was used to evaluate the myeloablative effect. RESULTS: 1) The optimal dose of busulfan was 100 mg/kg. The combination of busulfan and cyclophosphamide can restore the hematopoiesis of transplanted mice, and the toxicity associated with pretreatment is small. 2) In the allogeneic transplantation group: The hematopoietic reconstitution and high donor chimerism rate were achieved after transplantation. In the early phase of bone marrow transplantation, the T lymphocytes were the main cell group, while the recovery of B lymphocytes was relatively delayed. The dendritic cells and natural killer cells from donors were the earliest cells to recover and achieve high chimerism rate compared with T cells and B cells. Most T cells were in the initial T cell state within 5 days after allogeneic transplantation. However, in the 5th day after transplantation, these cells were mainly in the effective memory phenotype. The reconstruction of donor-derived naive T cells was slow, but the reconstruction of donor-derived effective memory T cells and regulatory T cells was relatively fast. 3) In the homogeneic transplantation group: The mice could recover hematopoiesis and the recovery of B lymphocytes was delayed. 4) In the chemotherapy alone group: All mice died in 12-15 days after chemotherapy, and the peripheral blood routine showed pancytopenia before death. CONCLUSIONS Pretreatment with chemotherapy can successfully establish the mouse model of bone marrow transplantation. There are difference in the proportion of T cells, B cells, natural killer cells, dendritic cells, effector memory T cells, initial T cells, and regulatory T cells after transplantation, and the relationship between donor and recipient is also changed.
Animals ; Bone Marrow Cells ; Bone Marrow Transplantation ; Busulfan ; Cell Proliferation ; Kinetics ; Mice ; Mice, Inbred C57BL ; Transplantation, Homologous

Objective:To analyze the DNA sequences and clinical phenotypes of four cases with rare thalassemia to improve its recognition and accurate diagnosis.Methods:The DNA sequence characteristics of four cases with rare thalassemia diagnosed from May 2014 to December 2019 were retrospectively analyzed, and related literature was reviewed.Results:The results of the routine gene test for thalassemia indicated that the common three type of deletion and three point mutations in hemoglobin alpha 1/2 (HBA1/A2) , and 16 point mutations in hemoglobin beta (HBB) gene were unable to be detected in cases 1-3, and case 4 was--SEA. However, the results of HBA1/A2 and HBB whole-genome sequencing revealed that the four cases had a point mutation of HBB:c.347C>A, HBB:c.1A>G, HBB:c.393T>G, and HBA2: c.301-1G>A (IVS II-142 G>A) , respectively. Meanwhile, the father, aunt, and grandfather of case 2 carried the HBB:c.1 A>G heterozygous point mutation.Conclusion:The novel mutations in HBB and HBA2 genes, resulting in a rare thalassemia, were revealed. Among them, the HBB:c.347C>A, HBB:c.1A>G, and HBA2:c.301-1G>A (IVS II-142 G>A) mutations were first reported in Chinese patients with thalassemia. Contrarily, HBB:c.393T>G mutation has not yet been recorded in the databases of human hemoglobin variants and thalassemia. The discovery of these novel nucleotide variants in this study would enrich the DNA mutation gene database of thalassemia.