| ObjectiveTo investigate the curative effect of combination therapy with As203 and ART o n acute promyelocytic leukemia(APL)by establishing animal model of APL.This study aims at exploring the action mechanism of ART by observing the effect of combination with As203 and ART on the growth inhibition,cycle distribution and the expression of apoptosis related proteins using acute promyelocytic leukemia NB4 cell as the research object.Methods1.Establish animal model of acute promyelocytic leukemiaFVB mice,6-8 weeks old,were divided into two groups:one group was injected with spleen cells carrying PML-RAR and GFP gene into tail vein,the other group was control group without any treatment.The proportion of GFP in peripheral blood was detected by Flow cytometry,pathological sections were taken from the main tissues,and RNA was extracted from splenocytes for gene expression by PCR analysis.2.Study on pharmacodynamics of combination of arsenic trioxide and artesunate based on FVB mouse modelModeling:model of FVB mice was injected with tumor cells into caudal vein.The mice were randomly divided into eight groups:the normal control group,the model group,the RA group,the ATO+ART group,the RA+ART group,the ATO+RA+ART group.The survival time of mice,the positive rate of GFP in peripheral blood,the positive rate of bone marrow cell surface antigen(CD34/CD117),and the pathological examination of main organs were studied.3.Inhibitory effect of combination of arsenic trioxide and artesunate on proliferation of NB4 cellsThe inhibitory effects of different doses of arsenic trioxide,artesunate and their combination on the proliferation of NB4 cells were determined by MTT assay,and median inhibitory concentration was calculated.4.Effects of combination of arsenic trioxide and artesunate on proliferation and apoptosis of NB4 cellsNB4 cells were treated with arsenic trioxide,artesunate and combination of them.The cell proliferation cycle distribution and apoptosis were detected by flow cytometry.5.Effects of combination of arsenic trioxide and artesunate on proteins related to NB4 cellsNB4 cells were treated with arsenic trioxide,artesunate and the combination of them.Western blot was used to analyze the expression levels of NF-κB,VEGF,Survivin and MMP-9.Result1.The FVB-APL mice model was well established by the mentioned ways.15 days after inoculation s,GFP could be detected in the peripheral blood of mice.22 days later,a performance of mental burnout,less activity,disorderly back hair,decreased food intake,fecal sugar thinning and beginning to die,and the dead mice began to bleed from mouth and nose.These performances accord with the clinical hemorrhage symptom of acute promyelocytic leukemia.Pathological examination showed that the important organs of mice had different degree of damage,and pathogenic genes could be detected by PCR.2.Study on pharmacodynamics of combination of arsenic trioxide and artesunate based on FVB mouse modelAfter treatment with arsenic trioxide and artesunate,the survival time of mice were prolonged(P<0.05),and the GFP in peripheral blood was decreased(P<0.05).The total bone marrow cells differentiation was significantly improved compared with the model group(P<0.05),and the pathological examination showed that the injury of main organs was obviously improved in the administration group.3.Inhibitory effect of combination of arsenic trioxide and artesunate on proliferation of NB4 cellsBoth arsenic trioxide and artesunate showed inhibition on the proliferation of NB4 cells in a dose-dependent manner.Combination of them showed better effect than that of them alone(P<0.05).4.Effects of combination of arsenic trioxide and artesunate on proliferation and apoptosis of NB4 cellsBoth arsenic trioxide and artesunate could significantly block the proliferation cycle and induce apoptosis of NB4 cells(P<0.05).Combination of them showed better effect than that of them alone(P<0.05).5.Effects of combination of arsenic trioxide and artesunate on proteins related to NB4 cellsBoth arsenic trioxide and artesunate showed significantly down-regulation on the expression levels of NF-κB,VEGF,Survivin and MMP-9 in NB4 cells.Conclusion1.Acute promyelocytic leukemia model can be successfully established by tail vein injection of spleen cells carrying genes in FVB mice.This model is a commonly used model to study the development and treatment of acute promyelocytic leukemia at home and abroad.2.Artesunate can enhance the therapeutic effect of arsenic trioxide and retinoic acid in the treatment of acute promyelocytic leukemia,delay the disease process,prolong the survival time,and provide experimental basis for new clinical medication.3.Artesunate alone could inhibit the proliferation of NB4 cells in a dose-dependent manner.4.Inhibiting cell proliferation and inducing apoptosis is one of the possible mechanisms of arsenic trioxide alone combined with artesunate in inhibiting proliferation of NB4 cells.5.Inhibition of tumor proliferation and expression of metastasis related proteins may be one of the mechanisms of arsenic trioxide combined with artesunate to improve acute promyelocytic leukemia. |
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