Wang Shu-Jin, Yu Xiang-Ru, Zhang Qi-Gang, Li Yan-Jie, Fu Chun-Ling, Xu Kai-Lin
Blood Diseases Institute, Xuzhou Medical University; Jiangsu Key Laboratory of Bone Marrow Stem Cells;Xuzhou 221000, Jiangsu Province, China.
Blood Diseases Institute, Xuzhou Medical University; Jiangsu Key Laboratory of Bone Marrow Stem Cells; Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou 221000, Jiangsu Province, China.E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2023 Aug;31(4):1113-1118. doi: 10.19746/j.cnki.issn.1009-2137.2023.04.028.
To construct a myeloproliferative neoplasms (MPN) transplanted mouse model with or gene mutation, and establish a systematic evaluation system to verify the success of model construction.
The bone marrow c-kit cells of the mice were obtained by the following steps: The mice were killed by cervical dislocation, the femur, tibia and ilium were separated, and the bone marrow cells were collected. The c-kit cells were sorted after incubation with CD117 magnetic beads. The method of constructing mouse primary mutant cells is as follows: A gene mutation vector with a GFP tag was constructed by the retroviral system, and the retroviral vector was packaged into the Platinum-E cells to obtain the virus supernatant, and then used it to infect the c-kit cells of mice. The MPN mouse model was constructed as follows: the mouse primary c-kit cells containing the mutant genes were collected after infection, and then transplanted them via the tail vein into the female recipient mice of the same species which were irradiated with a lethal dose of gamma rays (8.0 Gy). The MPN mouse model was evaluated as follows: After transplantation, the peripheral blood of the mice was regularly collected from the tail vein to perform the complete blood count test, and the size of spleen and the degree of bone marrow fibrosis were estimated.
The mouse c-kit cells with the mutant genes were successfully obtained from the bone marrow. MPN mouse model was successfully constructed: The peripheral blood cells of the MPN-transplanted mice carried exogenous implanted GFP-positive cells, and the white blood cells (WBC), platelet (PLT) and hematocrit (HCT) were all increased; the body weight loss, and the water and food intake were reduced in the transplanted mice; further pathological analysis showed that the transplanted mice displayed splenomegaly and bone marrow fibrosis. These results suggested that the MPN mouse model was successfully constructed. According to the common and different characteristics of the three MPN mouse model, a preliminary evaluation system for judging the success of MPN mouse model construction was summarized, which mainly included the following indicators, for example, the proportion of GFP-positive cells in the peripheral blood of mice; WBC, PLT and HCT; the degree of spleen enlargement and the bone marrow fibrosis.
The MPN mouse model with or gene mutation is successfully established by retroviral system, which can provide an important experimental animal model for the research of MPN pathogenesis and drug-targeted therapy.
构建携带 或 基因突变的骨髓增殖性肿瘤（MPN）移植小鼠模型，并建立系统的评估体系以验证模型构建是否成功。
通过以下步骤获取小鼠骨髓c-kit细胞：颈椎脱臼处死小鼠，分离股骨、胫骨和髂骨，收集骨髓细胞。用CD117磁珠孵育后分选c-kit细胞。构建小鼠原代突变细胞的方法如下：利用逆转录病毒系统构建带有绿色荧光蛋白（GFP）标签的基因突变载体，将逆转录病毒载体包装到Platinum-E细胞中获得病毒上清液，然后用其感染小鼠的c-kit细胞。构建MPN小鼠模型的方法如下：感染后收集含有突变基因的小鼠原代c-kit细胞，然后通过尾静脉将其移植到经致死剂量γ射线（8.0 Gy）照射的同品系雌性受体小鼠体内。对MPN小鼠模型进行如下评估：移植后，定期从小鼠尾静脉采集外周血进行全血细胞计数检测，并评估脾脏大小和骨髓纤维化程度。
成功从骨髓中获得携带突变基因的小鼠c-kit细胞。成功构建了MPN小鼠模型：MPN移植小鼠的外周血细胞携带外源植入的GFP阳性细胞，白细胞（WBC）、血小板（PLT）和血细胞比容（HCT）均升高；移植小鼠体重减轻，水和食物摄入量减少；进一步的病理分析表明，移植小鼠出现脾肿大和骨髓纤维化。这些结果表明成功构建了MPN小鼠模型。根据三种MPN小鼠模型的共同和不同特征，总结了判断MPN小鼠模型构建成功的初步评估体系，主要包括以下指标，例如，小鼠外周血中GFP阳性细胞的比例；WBC、PLT和HCT；脾脏肿大程度和骨髓纤维化程度。
利用逆转录病毒系统成功建立了携带 或 基因突变的MPN小鼠模型，可为MPN发病机制及药物靶向治疗的研究提供重要的实验动物模型。
