| Objective: To sort and identify leukemia stem cell (LSC) in childhood acute lymphoblastic leukemia (ALL), to establish LSC- leukemia model in NOD-SCID mouse to investigate the effects of LSC on the survival, to observe the association of LSC with ALL clinical characteristics, to further explore the expressions ofβ-arrestin1 and related genes, to illustrate the pathogenetic mechanism and provide potential therapeutic targets of childhood ALL.Methods: The assays, including magnetic activated cell sorting (MACS), flow cytometry, RT-PCR and FISH, were utilized to identify LSC in childhood ALL. And then LSC cell population was injected into the tail vein of irradiated NOD-SCID mice to establish leukemia model. Expression of CD34~+CD38~-, the surface phenotype of LSC, was analyzed by flow cytometry to investigate its association with clinical characteristics and prognosis of ALL. The role of LSC on the prognosis for childhood ALL was validated by leukemia mouse model. Furthermore, the mRNA and protein expression ofβ-arrestin1 was detected by real-time PCR, immunofluorescence and immunohistochemistry assays in isolated cells from ALL patients and bone marrow mononuclear (BMMN) from mice. The mRNA expression of other related genes was also measured by real-time RT-PCR.Results: The LSC (CD34~+CD38~- cell population) was successfully isolated by flow cytometry, in which cell population LSC related molecular marker CD19 and ALL fusion genes were expressed. After injecting CD34~+CD38~- cells into NOD-SCID mice, the WBC count was gradually elevated with blasts presented in peripheral blood and the ALL image phenotype of bone marrow was mimicked in the mouse, which indicated that leukemia mouse model was successfully established. Compared with mice in control group and CD34-CD38+ group, the total survival of mice with CD34~+CD38~- group was lowest (P<0.05). Moreover, we found that CD34~+CD38~- expression was positively associated with the ALL prognostic factors, such as age, peripheral blood WBC, peripheral blood blast cell count, cytogenetics, ALL fusion gene, early chemotherapy response, extramedullary infiltration and risk stratification (P<0.05) by analyzing clinical cases. In addition, the mRNA and protein expressions ofβ-arrestin1 in CD34~+CD38~- cells were increased compared with controls (P<0.05). Both the mRNA expression of Wnt3a andβ-catenin was also increased (P<0.05).Conclusion: The childhood acute lymphoblastic leukemia stem cell was successfully isolated and identified. NOD-SCID leukemia mouse model was successfully established. The total survival rate of mice with CD34~+CD38~- cells was significantly decreased. The high expression of CD34~+/CD38~- was positively correlated with poor prognostic factors of childhood ALL patients. Furthermore, the expression ofβ-arrestin1, Wnt3a andβ-catenin was increased in CD34~+CD38~- cells.
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