Construction Of The Bcr/abl Retroviral Vector-Mediated Murine Model Of Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal proliferating disease originating from the multipotent hematopoietic stem cells and the persistent expression of the bcr/abl fusion gene plays an essential role in the pathogenesis of CML. Nowadays, in vitro cell models containing the bcr/abl fusion gene were employed to explore the pathogenesis and treatment strategy in our country, and the propagation of bcr/abl transformed hematopoietic cell lines in mice were used as an in vivo murine CML model. Only recently, was the strategy of xenotransplantation of human K562 CML cells into immunodeficient mice first reported after an adaptation of the K562 cells in the subcutaneous neoplasm of the ethylic mouse. Retroviral bone marrow transduction/transplantation murine model of bcr/abl induced leukemia was employed to explore the genesis, development and bionomics of human CML overseas ever sine 1990s'and it is now the most efficient murine CML model able to induce the human MPD like disease. However, there is yet no report on the successful construction of the above murine CML model in our country. The present study has successfully constructed the CML murine model taking the Mig210 retroviral vector containing the bcr/abl fusion gene as a vector, which was subsequently packaged and the resultant retroviral supernatant was collected and used to infect the bone marrow cells from the 5-FU primed male Balb/c mouse after the detection of its titer. The infected donor bone marrow cells were then injected into the homogenous female Balb/c recipent mouse pretreated byγradiation at a lethal dose. Meanwhile, the present study have undertaken a series of optimizations on the basis of the previous experiences in the construction of the CML murine model and had successfully set up the secondary transplantation model. The main methods, results and conclusions were represented as follows: 1.The construction of the bcr-abl retroviral vector-mediated murine model of chronic myeloid leukemia.The Mig210 retroviral vector containing the bcr-abl fusion gene was packaged by Phoenix-Ampo cells, and the retroviral supernatant, which was then used to infect the bone marrow cells from the 5-FU-primed male Balb/c donor mouse, was collected. After being infected by the retrovirus, the donor bone marrow cells were transplanted into the lethally irradiated (900cGyγ-ray) homogenous female recipient mouse intravenously via its vena caudalis. Morphological observation of the bone marrow cells, RT-PCR and western blot respectively detecting the mRNA and protein expression level of the bcr/abl fusion gene were employed to evaluate the murine CML model system. After 8-9 weeks'transplantation, the white blood cells in the peripheral blood significantly increased to 20~30×109/L (5-8 times that of the normal control mice); and the proportion of the blast cells in the blood smear reached 10%~20%; Results from bone marrow slides showed that the cell population of the granulocytic lineage also increased significantly and the blast cells were readily found; What's more, the leukemic cells were found to be infiltrating into the liver and spleen of the candidate model mouse. The bcr-abl fusion gene was detected by RT-PCR in the bone marrow and spleen within 4~5 weeks'time, and in the liver within 8~9 weeks from the recipient mouse, and at the same time, the BCR-ABL fusion protein was detected in the bone marrow and spleen as shown by the results from western blot. The mice in which the CML model were successfully constructed died one after another in 3~5 months'time. The experiments were optimized and improvements were made as follows: The bone marrow cells were infected by the retroviral supernatant on fibronectin and the live cells were shown to be increased by 20%~30% when compared with the ones infected without the fibronectin; The preconditioning doses of 5-FU used to treat the male donor Balb/c mice were grouped into 2mg, 5mg and 10mg per 10g murine weight and after 4 days'treatment the bone marrow cells were collected to calculate the relative number of the blast cells. It was found that in a certain range, the dosage of 5-FU didn't influence the relative number of the blast cells significantly; When the bone marrow cells injected into the recipient mice were grouped into 0.2×106,0.4×106,0.6×106 and 1.0×106, the corresponding rate for successful model construction was respectively 60%, 80%, 90% and 90%.2. Construction of the secondary transplantation murine model for chronic myeloid leukemia mediated by bcr-abl retroviral vector.The bone marrow cells or spleen cells from the primary animals in which the bcr-abl retroviral vector-mediated murine chronic myeloid leukemia model had previously been successfully established were collected and subsequently injected intravenously into the sublethally irradiated (450 cGyγ-ray) female Balb/c recipient mice. Spleen colony transformation assay, morphological observation of the bone marrow, detection of the Y chromosome and its specific sex determining gene Sry and the analysis of the expression level of bcr/abl fusion gene both at its mRNA and protein level were employed to evaluate the adoptive transfer of the CML-like syndrome in the murine secondary transplantation model. The day 12 spleen colonies were visible in appearance and bulk of spleen colonies could be seen in serial sections; The probable Y chromosome and its specific Sry gene were detected within 4~5 weeks'time in bone marrow cells from the female recipient mice, and at the same time, the bcr-abl fusion gene was detected in the bone marrow and spleen; After 6-8 weeks'transplantation, the white blood cells in the peripheral blood were significantly increased by 4-8 times that of the normal control mice ,with granulocytes reaching 20~30×109/L; the proportion of the blast cells in the blood smear reached 10%~20%; Results from bone marrow slides showed that the cell population of the granulocytic lineage also increased significantly and the blast cells were readily found; What's more, the bcr-abl fusion gene was detected in the liver and the BCR-ABL fusion protein was detected in the bone marrow. Meanwhile, the leukemic cells were found to be infiltrating into the liver and spleen of the candidate secondary transplantation model mice, which all succumbed to blast crisis within 18 weeks'time and eventually died one after another due to lung hemorrhage.Taken together, the bcr-abl retroviral vector-mediated murine model of chronic myeloid leukemia was successfully constructed for the first time in our country, and a series of optimizations were made. Meanwhile, the secondary murine transplantation model was successfully set up, which could be used in the subsequent researches on CML in the fields of cell signal transductions and therapeutic insults.