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.
目的: 通过化学治疗(以下简称化疗)药物预处理建立小鼠骨髓移植模型，探讨异基因移植早期小鼠脾各免疫细胞的动态变化。方法: 小鼠按白消安剂量的差异分为4个组(80 mg/kg组、100 mg/kg组、120 mg/kg组、150 mg/kg组)，并与环磷酰胺进行联合化疗，通过评估清髓效果和药物毒性来确定合适用药剂量。实验按基因移植类型分为4组：异基因移植组、同基因移植组、单纯化疗组和正常对照组。使用白消安和环磷酰胺对移植小鼠化疗预处理后进行骨髓移植。异基因移植组在移植后第1天、第3天、第5天和第8天4个时间点制备脾细胞悬液，进行流式细胞学检测，分析脾各免疫细胞的动态变化，并与同基因移植组进行同期对照，与正常对照组进行脾免疫细胞群基础值对照，通过单纯化疗组对清髓效果进行对照评估。结果: 1)白消安剂量100 mg/kg为最合适剂量，该剂量与环磷酰胺联合化疗能恢复移植小鼠造血且预处理的相关毒性较小。2)异基因移植组移植后能使造血重建达到高供者嵌合，T淋巴细胞为主要的细胞群体，B淋巴细胞恢复相对延迟。供者来源的树突状细胞和自然杀伤细胞为最早开始恢复并达较高嵌合率的细胞群体。异基因移植组移植后5 d内，大多数T细胞无明显表型，为初始T细胞状态，异基因移植5 d以后，这些细胞主要是效应性记忆表型。供者来源的初始T细胞重建缓慢，但供者来源的效应性记忆T细胞和调节T细胞重建相对较快。3)同基因移植组移植后小鼠能恢复，B淋巴细胞恢复延迟。4)单纯化疗组小鼠在化疗后的第12~15天全部死亡，死亡前外周血常规检查均提示为全血细胞减少。结论: 使用化疗药物进行预处理能成功建立小鼠骨髓移植模型。T细胞、B细胞、自然杀伤细胞、树突状细胞、效应性记忆T细胞、初始T细胞及调节T细胞在异基因移植后的比例及供受比关系均有不同程度的变化。.
Keywords: hematopoietic stem cell transplantation; immunity; mouse model.