| Objective and Significiance:Acute leukemia is a kind of malignant clone hematopoietic desease with features of high heterogeneity and mortality.The data of existing clinical researches shows that nearly 8%of the AML patients can be detected highly expressed EVI1 gene,indicating a poor prognosis and resistance to chemotherapy.The chromosome of 3q26 where EVI1 gene locates at is prone to break and form the t(3;21)(q26;q22)Runx1-Evi-1(AML1-EVI1)fusion gene which can accelerate the process of the occurrence and development of leukemia through various of pathways.Little was known about the effection of EVI1 gene on hematopoiesis,though there are many scholars being committed to the study.Futhermore,almost no research concerning the difference of the function among EVI1,AML1 and the fusion gene of AML1-EVI1 can be found till now.Thus,this research takes advantage of the cell line from AML patients and transgenetic zebrafish to exam the hematopoietic regulation factors,of the genes mentioned before in vitro and vivo and evaluate the effects induced by arsenic trioxide(ATO).Finally,this research has established a zebrafish model for evaluating the cardiotoxicity induced by Doxorubicin(DOX),which is the most commonly used chemotherapeutic agnet,and mechanism of the protective effect of Dexrazoxane(DZR)on account of the interesting discovery during the experimentsMethod:The cell line of THP1 which has a high EVI1 expression from acute myeloid leukemia patients were chosen to evaluate the mRNA relative expression of EVI1 gene and hematopoietic transcription factors like GATA1,GATA2,RUNX1,MPO,LM02,CMYB,PU.1 and SCL through reverse transcription-real-time fluorescent quantitative polymerase chain reaction(RFQ-PCR)in vitro.Healthy adult peripheral blood mononuclear cells were used as a control.After that,THP-1 cell line was treated with ATO solution with concentrations of 1 u M?3 u M?5 u M.Then the mRNA relative expression of EVI1 gene and hematopoietic transcription factors were detected by RFQ-PCR..Genes of AML1 and EVI1 from the fusion gene of human AML1-EVI1 were installed into the plasmid carrier of TOL2 with enhanced green fluorescence protein reporter gene respectively and microinjected into the zebrafish embryo in one cell state.Then the embryo of founders which can successfully express the target genes were selected out and raised to their sexual maturity.The F1 zebrafish which were generated by AB wild type and transgenetic zebrafish(Tol2-EVI1-EGFP:TE;Tol2-AML1-EGFP:TA)were verified to express the specific target genes at ldpf,3dpf and 7dpf.Embryo were dosed with ATO in a concentration of 50? M and 100?M when they developed to 18hpf,and the mRNA of EVI1 and hematopoietic transcription factors were tested at ldpf,3dpf and 7dpf seperately.The last part of this experiment,we chose the AB strain embryo of 24hpf,and put them into the solution of DOX with concentrations of 0,1.84 u M,18.40 u M,36.80?M,46.00 ?M,55.20 u M,64.40 u M,73.60 u M,82.79 ?M and 91.99 ?M,finding that the DOX with concentration of 64.40 ?M can induce the most significant phenotype.So we also pretreated the embryo with DZR for 48h.The concentration of DZR is 130.47 ?M and 260.93 ?M.As the zebrafish developed to 72hpf,the morphological variation of cardiovascular system were observed through microscope and the changes of heart rate were recorded.In addition,the whole RNA of zebrafish was extracted and the heart development related genes and index of oxidation and anti-oxidation process were also detected.Result:THP1 cell line with high expression of EVI1 gene was selected to comfirm that EVI1 can up regulate the expression of GATA2 and CMYB,and reduce the level of GATA1?RUNX1?MPO?LM02?PU.1 and SCL simultaneously in vitro.Moreover,ATO has the ability to down regulate EVI1 gene in a dose-dependent and time-dependent maner,while the same effect of reduction can also be observed in other hematopoietic transcription factors.The transgenic zebrafish strains of TE and TA with high expression of human EVI1 and truncated AML1 were established and raised into F1 generation in success.We have substantiated that zebrafish with high expression of EVI1 tend to have severe phenotypes which also show deficiency of hematopoietic transcription factors in different levels.And TE zebrafish with treatment of ATO have shown the reduction of EVI1 expression,the high expression of GATA2 induced by EVI1 can also be down regulated meanwhile.Along with the concentration of DOX increases,kinds of malformation like developmental malformation and pericardium edema have occurred among zebrafish embryos with an ascending rate of mortality.Dosing DZR with DOX can markedly help to reduce the cardiotoxicity caused by DOX and significantly enhance embryos' survival rate.The survival rate of zebrafish embryos can be improved to 90%and 88.3%from 30%by adding 130.47 u M or 260.93 u M DZR separately into DOX solution,p<0.001.The results of RT-PCR examining the expression of some heart development relating genes such as nkx2.5,cmlc2,amhc and vmhc have shown the irrelevance between cardiotoxicity and the heart development at the genetic level.We examined the MDA level and SOD activity,which are two indicators of oxidation and anti-oxidation,in zebrafish embryos,and we have found DOX of 66.40?M can lift the MDA level and decrease the activity of SOD dramatically,which can be reversed largely by DZR(p<0.001).Conclusion:THP1 cell line and the new established transgenic zebrafish model with high expression of EVI1 gene were both used for experiments in vitro and in vivo.EVI1 gene has been substantiated to increase the expression of GATA2 transcription factor and exert different influences on other transcription factors to promote the proliferation of immature cells and inhibite the differentiation and devlopement of myelocytes and erythroid cells indirectly.Besides,ATO has shown the ability to down regulate EVI1 gene in specific way and decrese the activation of GATA2.The zebrafish model for assessing the cardiotoxicity induced by drugs. |
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