| During the development and differentiation of mammalian erythroid cells, the changes in cell morphology and gene expression showed regularity. The terminally differentiated cells are characterized by gradual narrowing of the cells and enucleation, and ultimately differentiating into mature red blood cells.when differentiations are hindered, the cells proliferate abnormally and cann’t express hemoglobin, then become erythroleukemia cells.It was once thought that normal cells form tumor cells is irreversible. Many studies found that tumor cells treated by the differentiation inducing agents might regain its differentiation capacity, it’s called induced differentiation. MEL cells were treated by the commonly used differentiation inducing agents, including dimethyl sulfoxide (DMSO),hexamethylene bisacetamide (HMBA),and sodium butyrate (NaBu) cells might regain its differentiation capacity and undergo normal differentiation. So erythroblasts provide a good model for the study of cell differentiation and dedifferentiation.The mouse were injected via the tail vein after FVA-induced anemia virus (FVA), erythroblasts were synchronized block in the original stage of immature red blood cells, under the simultaneous effect of Erythropoietin (EPO), the immature red blood cells can access to terminal differentiation in vitro. FVA cells retain the differentiation characteristics of CFU-E erythroid cells, which are similar to the differentiation process of normal erythroid cells, this model is used to study the differentiation of erythroid cells and leukemia pathogenesis. For understanding the mechanisms of erythroid cell differentiation and carcinogenesis, three aspects studies were carried out, which was based on the establishment of the models about differentiation of FVA cells and DMSO-induced MEL cells.1. Studying of the time-dependent morphological alteration during the differentiation of FVA CellsIn this dissertation, synchronized proerythroblasts induced by FVA virus transfection were used as a model. By using morphological staining, cell counting, real-time cultured cell monitoring for enucleation process, scanning electron microscopy (SEM) and immunofluorescence, combined with laser scanning confocal microscopy (LSCM), the FVA cells induced by EPO for0h,2h,12h,24h,36h,48h,60h,72h were used for:1) to study the shape and proportion of cells at different differentiation stages, enucleation process, the formation of blood islands, and engulfment of nuclei by macrophages, in real time;2) to quantitatively analyzing erythroid cell surface markers CD71and Ter119, and erythroid cytoskeletal-associated proteins (Stathmin, Septin8and RBBP4). In vitro, the cell volume gradually decreased. The results of real-time monitoring of enucleation showed that it takes about7to8hours from karyopyknosis (polychromatic erythroblasts) to extrude the nuclei. Scanning electron microscopy showed a variety of shapes of reticulocytes. It further showed that the macrophages engulfed the expelled erythroid nuclei. In erythroid cell differentiation process, expressions of both the transferring receptors CD71and Terl19were higher than that of non-erythroid cells, whereas, cytoskeletal-associated proteins (Stathmin, Septin8and RBBP4) decreased gradually during erythroid differentiation.2. Comparative proteomic analysis of differentiation in FVA cells and DMSO-induced MEL cells1). Comparative proteomic analysis of differentiation in FVA cells:The FVA cells induced by EPO for0h,2h,12h,24h,36h,48h,60h,72h were for proteomic analysis.117proteins or protein subunits were successfully identified, and it could be divided into12categories, the most three parts were32%enzyme,13%structural protein and10%regulatory protein.2). MEL cells were treated by DMSO, and the effects of the agent on cell differentiation and proliferation were detected by benzidine staining, MTT assay and Terl19immunofluorescence. And using two-dimensional gel electrophoresis combined with mass spectrometry technology and bioinformatics analysis, we conducted a comparative proteomic analysis on MEL cells during the process of induced differentiation to screen and identify differential proteins. MTT assay showed,1.0%,1.2%,!.4%DMSO showed no inhibiting effect on cell vitality. MEL cells exposed to DMSO at a final concentration of1.2%for120h reached the highest differentiation rate of67%. The MEL cells induced by1.2%DMSO for0h,6h,12h,24h,36h,48h,72h,96h,120h were for proteomic analysis. By two-dimensional gel electrophoresis combined with mass spectrometry,87proteins or protein subunits were successfully identified, and it could be divided into12categories, the most three parts were41%enzyme,15%structural protein and13%regulatory protein.There were28proteins both included in the differentially expressed proteins of DMSO-induced MEL cells and FVA cells, and it could be divided into8categories, It showed that these proteins play an important role in the differentiation process.3. A preliminary study of Stathmin in differentiationDuring the differentiation of FVA cells, there were many changes in cell morphology:karyopycnosis, enucleation and the obvious changes of cytoskeleton. The microtubule depolymerization protein Stathmin is a member of the protein family, which regulates the microtubule cytoskeleton. It also regulates microtubule polymerization via promoting microtubule depolymerization and preventing tubulin heterodimers.The Stathmin is a tumor marker protein, and the results of Western Blot showed that the Stathmin expressed low in the differentiation of MEL cells. So we used RNAi technology to analyzed the function of cytoskeletal-associated protein Stathmin from FVA model. Stathmin RNAi plasmids were constructed and generating lentiviral particles, then infected MEL cell, Cells that stably express shRNA were selected by puromycin. MTT assay showed, shRNA mediated knockdown of Stathmin protein showed promotion effect on cell vitality, which provides an experimental evidence for further investigating its role in the Erythroid cell differentiation. |
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