Objective: To construct a myeloproliferative neoplasms (MPN) transplanted mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation, and establish a systematic evaluation system to verify the success of model construction.
Methods: 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.
Results: 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.
Conclusion: The MPN mouse model with JAK2-V617F, MPLW515L or CALR-Type I 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.
题目: 骨髓增殖性肿瘤小鼠模型的构建及评价体系的建立.
目的: 构建携带JAK2-V617F、 MPLW515L及CALR-Type I基因突变的骨髓增殖性肿瘤(MPN)移植小鼠模型,并建立移植小鼠模型构建成功的评价体系。.
方法: 获取小鼠骨髓c-kit+细胞:颈椎脱位处死小鼠,分离股骨、胫骨及髂骨,收集骨髓细胞,经过CD117磁珠孵育,分选出c-kit+细胞。构建小鼠原代突变细胞:利用逆转录病毒体系构建GFP标记的基因突变载体,将带有基因突变的逆转录病毒载体经Platinum-E细胞包装后,获取病毒上清,感染小鼠的骨髓c-kit+细胞。构建MPN移植小鼠模型:收集感染后含有突变基因的小鼠原代c-kit+细胞,再经尾静脉植入经致死剂量(8.0 Gy)γ射线照射的同种雌性受鼠体内。评价MPN移植小鼠模型:移植后定期通过尾静脉采血检测小鼠外周血血象变 化;观察小鼠脾脏大小及骨髓纤维化的程度。.
结果: 成功获得携带突变基因的小鼠骨髓c-kit+细胞。成功构建MPN移植小鼠:移植小鼠外周血细胞中携带外源植入的GFP阳性细胞,且白细胞(WBC)、血小板(PLT)以及红细胞压积(HCT)均升高;移植小鼠重量减轻、饮水量与摄食量减少;病理分析显示移植小鼠脾脏肿大,并伴有骨髓纤维化,提示MPN模型构建成功。根据MPN模型建立过程中3种移植小鼠表现出的共性和异性,归纳总结出可以作为判定MPN模型构建成功的一个初步评价体系,主要包括以下指标:小鼠外周血GFP阳性细胞比例;WBC、PLT及HCT计数;脾脏肿大程度及骨髓纤维化程度。.
结论: 成功建立JAK2-V617F、 MPLW515L及CALR-Type I逆转录病毒介导的MPN小鼠模型,可为针对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。.
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