Study On The Establishment Of Animal Model Of Acute Myeloid Leukemia Induced By Benzene And Its Mechanism

Objective:Benzene is a widely used organic solvent,and it is also a serious environmental pollutant.During the process of production,benzene is mainly absorbed through the respiratory tract.Chronic benzene exposure mainly damages the hematopoietic system,which can cause the decrease of white blood cell count,aplastic anemia and leukemia.However,the pathogenesis of leukemia caused by benzene has not been fully elucidated,and there was no idea animal model so far.This study aims to establish an animal model of acute myeloid leukemia induced by benzene,and to find out the key genes and pathways involved in benzene-induced leukemia.Methods:1.Transplantation experiment:bone marrow cells obtained from a 2-month-old Mll-Af9+/-mouse were transplanted through tail vein into 2-month-old B6.SJL mice irradiated with isotope 60Co.After three weeks of bone marrow reconstitution,flow cytometry was performed to examine whether the transplantation was successful.2.Establishment of dynamic inhalation poisoning model:the mice after transplantation were randomly divided into control group(n=32)and benzene group(n=32).In benzene exposure group,the concentration of benzene was set at 1000mg/m3,8h/d,5d/week,the concentration of benzene in the cabinet was detected by gas chromatography;The control group mice were exposed to clean air.The end point of the experiment was the occurrence of leukemia.During the process of poisoning,the changes of hematological parameters and the proportion of the tumor precursor cells(CD45.2+ cells)in the peripheral blood of mice were monitored once a week.In the 1st,3rd,5th,7th,9th,and 13th weeks,one mouse of the benzene group and control group were sacrificed to compare the self-renewal ability of the tumor precursors cells and normal HSPCs.At the same time,the difference of the development of leukemia and the survival curve between control group and benzene group were also compared,which could provide a clue for the study of the mechanism of acute myeloid leukemia induced by benzene.3.RNA sequencing and bioinformatics analysis:Total RNA of bone marrow cells from the leukemia mice in both group were extracted,and the characteristic signal pathway caused by benzene was searched by bioinformatics analysis.Results:1.Hematological toxicity induced by benzene:WBC counts,RBC counts and Hgb content in peripheral blood of the control group mice had no obvious change at the beginning stage.In the stage of the outbreak of leukemia in mice,WBC counts in the peripheral blood increased gradually,RBC counts and Hgb content reduced,several weeks later,the mice died;In the first week,WBC counts,RBC counts and Hgb content decreased obviously in benzene exposure group mice,significantly lower than the control group(p<0.05).The self-renewal capacity of hematopoietic progenitor cells was also inhibited when WBC decreased.When leukemia occurred in mice,WBC counts increased rapidly,RBC counts and Hgb content decreased significant.2.Benzene exposure promoted the accumulation of leukemia precursor cells:After the reconstruction of bone marrow,the proportion of the tumor precursor cells(CD45.2+ cells)in peripheral blood gradually increased,but the mice in the benzene group were significantly higher than that in the control group(p<0.05),which indicated that benzene exposure promoted the accumulation of tumor precursor cells.3.pathological results:the results of bone marrow smear showed that both the control group and the benzene group had acute myelocytic leukemia.The pathological results of the spleen showed that the infiltration of leukmia cells in the benzene group was more serious,which suggesting that the malignant degree of leukaemia in the infected mice was higher.4.RNA sequencing:we choose preleukemic mice without the disease as normal control,analysis of the transcriptional data,we found that the benzene group and control group mice showed significant acute myeloid leukemia transcriptome features:acute myeloid leukemia,chronic myeloid leukemia,NF-kappaB,and PI3K-Akt tumor necrosis factor,cancer and MAPK signaling pathways were activated.Compared with the control group,the hematological malignancies,hematopoietic sternness and self renewal signaling pathways were activated in the exposure group.The IPA analysis showed that the upstream regulators in the exposure group were activated.Conclusions:1.benzene exposure changed the pathogenesis of leukemia in mice:benzene exposure first caused the decrease of peripheral white blood cell counts and the accumulation of tumor precursor cells,and the white blood cell count increased rapidly and died rapidly in the later stage,which was different from that in the control group.Therefore,benzene exposure plays a certain role in the pathogenesis of leukemia in mice,and the animal model of leukemia induced by benzene has been established successfully.2.benzene exposure increased the malignancy of leukemia in mice,and the significant activation of self-renewal signaling pathway may be involved in the process of leukemia induced by benzene.