原代Ph^(+)骨髓细胞的获取及小鼠Ph^(+)B-ALL模型的建立

Acquisition of Primary Ph^(+)Bone Marrow Cells and Establishment of Ph^(+)B-ALL Mouse Model

目的:制备小鼠费城染色体阳性(Ph^(+))原代细胞并构建B淋巴细胞白血病(B-ALL)小鼠模型。方法:利用逆转录病毒将带有BCR-ABL P210融合基因的质粒转入C57BL/6J小鼠骨髓细胞中,输注给9 Gy总致死剂量60Coγ射线照射的同系小鼠后建立第一代Ph^(+)骨髓细胞的小鼠模型,然后获取发病小鼠脾脏和骨髓的原代细胞冻存。C57BL/6J小鼠经亚致死剂量照射后,接受第一代Ph^(+)细胞进行体内传代,顺序传代获得第三、四代Ph^(+)细胞及小鼠B-ALL模型。分别应用流式细胞术、H&E染色、外周血涂片等对建模小鼠进行免疫表型分析及病变检测。结果:输注含有P210-NGFR逆转录病毒的骨髓细胞后,小鼠出现体重明显下降、双下肢瘫痪、弓背等症状。发病小鼠的外周血涂片中可观察到原始和幼稚淋巴细胞。H&E染色结果显示,发病小鼠肝脏小叶中央静脉周围以及肝脏边缘有明显的白血病细胞浸润。流式细胞术检测结果显示,发病小鼠脾脏中CD19+NGFR+细胞随传代增加百分率逐渐升高,G1、G2和G3分别为19.0%、47.3%和61.0%。免疫表型分析结果表明,Ph^(+)细胞在B淋巴细胞中稳定传代,且随着传代增多Ph^(+)B淋巴细胞占比显著提高。结论:本研究成功制备小鼠Ph^(+)原代细胞,并顺利完成体内传代及B-ALL小鼠模型构建。

Objective:To harvest the primary Philadelphia chromosome-positive(Ph^(+))cells of B-acute lymphoblastic leukemia(B-ALL)and to establish the B-ALL mouse model.Methods:The plasmid carrying BCR-ABL P210 fusion gene was transferred into the bone marrow(BM)cells of C57BL/6J mice by retrovirus.Syngeneic mice irradiated with 9 Gy of 60Coγ-ray were injected with the transfected BM cells as the first generation(G1),and then the primary cells from the spleen and BM of the diseased mice were obtained and frozen.Sublethalγ-ray irradiated C57BL/6J mice were inoculated with the first generation of Ph^(+)cells for in vivo passage,which were named as the second generation(G2).The third and fourth generations(G3 and G4)of Ph^(+)cells and B-ALL mouse model were established by successive passages.Flow cytometry,H&E staining and peripheral blood smear were used to analyze the immunophenotypes and detect the pathological changes of the model mice.Results:After infusion of P210-NGFR retrovirus infected BM cells,the mice exhibited significant symptoms including weight loss,lower limbs paralysis,and arched back.Primitive and immature lymphocytes were observed in peripheral blood smears of the leukemia mice.The results of H&E staining showed obvious infiltration of leukemic cells around the central vein of the hepatic lobule and at the edge of the liver in the diseased mice.The results of flow cytometry showed that the percentages of CD19+NGFR+cells in spleen of the model mice were gradually increased with passage,which was 19.0%,47.3%and 61.0%in G1,G2 and G3 mice,respectively.Immunophenotypic analysis indicated that Ph^(+)cells were stably passaged in B lymphocytes,and the purity of Ph^(+)B lymphocytes was obviously elevated with the increase of passage frequency.Conclusion:In the present study,the primary Ph^(+)cells were successfully obtatined and passaged in vivo,and the B-ALL mouse model was successfully established.