| BackgroundAcute myeloid leukemia (AML) is a kind of lethal disease originating from acute myeloid leukemia stem cells with the characteristics of incontrollable proliferation of myeloid cells. The clinical outcome of AML is related with a lot of factors such as myeloid cell type, cytogenetic change as well as the biological characteristics of clones. Although treatment of AML is developing really fast recent years with a better treatment efficacy being achieved, relapse and treatment related side effect still remain to be the main obstacle of the improvement of efficacy of AML treatment. Previous studies have demonstrated that chemotherapy can’t eliminate leukemia stem cells and thus remaining leukemia stem cells can cause the relapse of patients. So now researchers are focusing on the pathogenesis of leukemia stem cells to develop new drugs for treating AML.AML originates from a small number of cells named AML stem cells (LSCs) which are characterized by their ability to self-renew, unlimited repopulating potential and long residence in a quiescent state of G0/G1phase. AML stem cells have some common markers such as CD34+and CD38". They are more resistant to common chemo-drugs and have cross-resistance with a lot of drugs. Therefore, method to eliminate AML stem cells have become the hot point for leukemia researches.Although AML has many types, all other types of AML leukemia stem cells, apart from APL, share the same surface markers such as CD34+and CD38-. Previous studies focusing LSCs always select CD34+and CD38-cells from the bone marrow of patients as their experimental subjects. However, the resource is not enough for research. Thus, it requires a new resource to select LSCs. KGla cell line originates from a male patient who had AML-MO and was resistant to chemo-drugs. This cell line has higher expressions of MDR and P-glycoprotein and is highly resistant to common chemo-drugs. Previous studies have already select LSCs from KGla cells to do researches focusing on LSCs.The foundation for researches focusing on the drugs to eliminate LSCs is the establishment of animal models of LSCs. Luckily, one of the characteristics of LSCs is the ability to establish leukemia animal model by injecting LSCs into NOD/SCID mouse. NOD/SCID mouse were developed by crossing SCID mouse with nonobese diabetic (NOD/Lt) mouse. This kind of mice has many characteristics such as lack of NK cells as well as complements, dysfunction of differentiation of antigen presenting cells, and lower ability to induce autoimmunity diabetes. Thus it is suitable for transplantation of leukemia cells to establish animal model for malignant hematology cancers while avoiding graft versus host disease(GVHD). Therefore, we use NOD/SCID mouse and CD34+CD38-KG1a to establish LSCs animal model to build a reliable base for in vivo LSCs study.AimTo establish a LSCs animal model using NOD/SCID and CD34+CD38-KG1a cells while exploring into the biological characteristics of LSCs animal model to provide a reliable foundation for further study to selecting LSCs targeting experiments.Methods1. Flow cytometry was used to select CD34+CD38" cells from KG1a cells.2. NOD/SCID were randomized into control and experiment groups. Mouse in both groups received2Gy irradiation with X-ray and then mouse in experiment groups were injected intravenously with CD34+CD38-KGla (cell viability95%detected using Trypan blue exclusion method,2×106cells per mouse, total liquid volume:0.2ml) through tail vein while those in control group were given same amount of PBS.3. General condition of mouse was monitored. Body weight of mouse was recorded. Morphology of bone marrow and blood was also observed regularly.4. Mouse were sacrificed5weeks after transplantation with tissues of lung, spleen, liver, kidney and bone marrow being observed using histopathology method to detect the characteristics of leukemia.5. Karotype analysis was also done in female mouse who have leukemia.6. SPSS13.0was used for statistical analysis. Student’s t test and one way analysis of variance (ANOVA) followed by LSD or Dunnett T3Multiple Comparison Test were used to calculate the differences between the results. Data were expressed as mean±SD, and all the P values were given for two-sided tests, and P<0.05was considered as significantly changes.Results1. Before selection, the proportion of CD34+CD38" cells is (55.48±4.95)%while after selection by flow cytometry, the proportion rose to (93.16±2.7)%.2.4weeks after transplantation, mouse in experiment group were dispirited with low appetite, down-bent gait, wrinkled fur, slow move, intention to be crowded and no increase of weight.5weeks after, significant difference of weight was observed between2groups (P<0.05). Moreover, leukemia cells could be seen in blood or bone marrow smear.3.5weeks after transplantation, solid tumor could be observed in peritoneal cavity of mouse in experiment group with slightly larger spleen and liver than those of mouse in control group. Diffused tumor nodules and granular appearance could also been observed in spleen and liver of mouse in experiment group. Histopathology examination also showed that leukemia cells infiltrate liver, spleen, lung, kidney and bone marrow with the most significant infiltration being observed in bone marrow and spleen. Immunohistochemistry examination also showed that leukemia cells in spleen and liver will have human specific antigen CD45and CD34(Antibody used here was demonstrated to have no cross-reaction with cells of mouse).4. Karotype analysis showed that human karotype could be detected in bone marrow from mouse with abnormal karotype such as7q-,8p+,12p+,i(llq) and Y chromsome which are all characteristics of KG1a.ConclusionLSCs animal model was successfully being established using NOD/SCID and CD34+CD38-KGla which laid a solid foundation for study on the drugs to eliminate LSCs. |
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