[Acquisition of Primary Ph⁺ Bone Marrow Cells and Establishment of Ph⁺ B-ALL Mouse Model]

To harvest the primary Philadelphia chromosome-positive (Ph⁺) cells of B-acute lymphoblastic leukemia (B-ALL) and to establish the B-ALL mouse model.

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 ⁶⁰Co γ-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. Sublethal γ-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.

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.

In the present study, the primary Ph⁺ cells were successfully obtatined and passaged in vivo, and the B-ALL mouse model was successfully established.