Localization And Expression Of PML(NLS~-) Protein In NB4Cells And Transplanted Tumors In Nude Mice And Its Clinical Application

PART ⅠLOCALIZATION AND EXPRESSION OF PML(NLSˉ)PROTEIN IN NB4CELLSObjective: To verify the existence and location of PML（NLS-）proteinin NB4cells.Methods: The plasmid pCMV-HA-NE was electroporated into NB4cells, then NE protein was verified by western blot assay. Extracted thecytoplasm protein of NB4-HA-NE cells, NB4-HA cells, NB4cells, K562cells and peripheral neutrophils from healthy subjects and detected thePML（NLS-）protein by western blot assay. Immunofluorescence assay andconfocal laser microscopy were performed to localize the PML（NLS-）protein of these cells.Results: NE protein was detected by western blot assay in the NB4-HA-NE cells and PML（NLS-）protein was verified in the NB4-HA-NEcells by western blot assay, which demonstrated PML-RARα was cutsuccessfully by NE enzyme. The expression of PML（NLS-）protein almost wasn’t in the cytoplasm of other types of cells, while PML protein wasmainly in the nucleus.The results of immunofluorescence assay andconfocal laser microscopy indicated that the PML（NLS-）protein waslocalized in the cytoplasm of NB4-HA-NE cells, while PML protein wasalmost localized in the nucleus of other types of cells.Conclusion: PML（NLS-）protein was localized in the cytoplasm ofNB4-HA-NE cells using western blot assay, immunofluorescenceassay,confocal laser microscopy. PART ⅡLOCALIZATION AND EXPRESSION OF PML(NLSˉ)PROTEIN IN TRANSPLANTED TUMORS OF NUDEMICEObjective: To verify the existence and location of PML（NLS-）proteinin transplanted tumors of nude mice.Methods: NB4cells, K562cells and NB4-HA-NE cells which wereinoculated at the right back of nude mice were expressed in xenograft.Extracted the cytoplasm protein and nucleoprotein and detected thePML(NLS-) protein by western blot assay. Immunofluorescence assay and confocal laser microscopy were performed to localize the PML(NLS-)protein.Results: NB4cells, K562cells and NB4-HA-NE cells weresuccessfully injected in subcutaneous of nude mice. PML（NLS-）protein innude mice was expressed and localized in the cytoplasm of NB4-HA-NEcells by western blot, immunofluorescence assay, confocal lasermicroscopy and immunohistochemistry, while PML protein was almostlocalized in the nucleus of NB4and K562cells in nude mice.Conclusion: PML（NLS-）protein was localized in the cytoplasm ofNB4-HA-NE cells of transplanted tumors in nude mice using western blotassay, immunofluorescence assay,confocal laser microscopy andimmunohistochemistry. PART ШLOCALIZATION AND EXPRESSION OF PML(NLSˉ)PROTEIN IN SAMPLES COLLECTED FROM PATIENTSObjective: To verify the existence and location of PML（NLS-）proteinin samples collected from APL patients.Methods: Human peripheral neutrophils were isolated from M3,nonM3patients and healthy subjects. Western blot assay, immunofluorescence assay and confocal laser microscopy were used todetect PML（NLS-）protein in neutrophils.Results: PML（NLS-）protein was expressed and localized in thecytoplasm of neutrophils in M3manifested by Western blot,immunofluorescence assay and confocal laser microscopy, whilePML(NLSˉ) protein was undetectable in neutrophils from nonM3samplesand healthy subjects and PML protein was nearly localized in the nucleus.Conclusion: PML(NLSˉ) protein was detectable in the cytoplasm ofneutrophils from APL patients by Western blot assay, immunofluorescenceassay,and confocal laser microscopy.