A Comparative Proteomic Analysis Of Induced Erythroid Differentiation In Murine Erythroleukemia Cells

Leukemia is a common malignant blood disease.To date,there is no clear measure for prevention and treatment of leukemia at home and abroad.Although therapy of induced differentiation for leukemia has achieved significant clinical effectiveness and research work has confirmed that a variety of differentiation inducing agents can induce reversal of malignant cancer cells to the erythroid,myeloid or other directions of differentiation,its mechanism is not fully understood.The induced differentiation of leukemia has become one of the hottest research areas of cell differentiation and carcinogenesis.Cell proliferation and differentiation is eo-regulated by a series of genes and proteins,and proteomic analysis can uncover the cellular network of proteins and its changes in a large scale.Therefore,comparative proteomic studies on induced differentiation of erythroleukemia cells will have important theoretical value for the clarification of molecular mechanism involved in erythroid leukemia cell differentiation and clinical value for the development of targeted therapy in erythroleukemia-specific drugs,and also provide experimental evidence for the search and improvement of erythroid differentiation-related regulatory network.In this thesis,murine erythroleukemia(MEL)cells were treated by three commonly used differentiation inducing agents,including dimethyl sulfoxide(DMSO),sodium butyrate(NaBu)and hexamethylene bisacetamide(HMBA),and the effects of these agents on cell proliferation and differentiation before and after treatment were detected by measuring cell growth rate,MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay and benzidine staining.Subsequently,by comparison,we selected the agent with the highest inducing rate to treat MEL cells,and using two-dimensional electrophoresis combined with mass spectrometry technology and bioinformatics analysis,we conducted a comparative proteomic analysis on MEL cells before and after induction to screen and identify erythroid differentiation-related proteins.Results showed that three inducing agents all showed inhibiting effect on cell proliferation and vitality.After exposed to a series of certain concentrations of the three inducing agents for a certain time,MEL cells were induced to differentiate with a high efficiency(>60%).Among these agents,MEL cells exposed to NaBu at a final concentration of 1.25 mM for 96 h reached the highest differentiation rate of 81%.Therefore we selected 1.25 mM NaBu to treat MEL cells for proteomic analysis.By two-dimensional gel electrophoresis combined with mass spectrometry,35 proteins or protein subunits were successfully identified,including 3 hemoglobin subunits and 11 histone subunits.Other differentially-expressed proteins are involved in the synthesis of hemoglobin,gene transcription and mRNA processing,protein translation and post-translation modification,cell division,cell immigration and the degradation of biomacromolecules.Besides,1 protein whose function is unknown was identified.These results may help to further clarify the molecular mechanism involved in NaBu-induced erythroid differentiation of MEL cells.