Location Verification Of NLS-RARα Protein In NB4Cells Electroporated PCMV-HA-NE Plasmid And Preliminary Application

PARTⅠ Location Verification of NLS-RARα Protein in NB4cellselectroporated pCMV-HA-NE plasmidObjective: To inverify the presence and location of NLS-RARαprotein in NB4cells electroporated pCMV-HA-NE plasmid.Methods: Electroporated pCMV-HA-NE plasmid into NB4cells.RT-PCR and Western blot were used to identify the expressed success inmRNA level and protein level in electroporated cells; Extracted thenucleoprotein from successful electroporated NB4cells, using Western blotmethod to determine the expression of NLS-RARAα protein inelectroporated NB4cells nucleus; FITC-AnnexinV/DAPI double stainingimmunofluorescence and confocal laser scanning method were used toexamine NLS-RARAα protein expression and localization inelectroporated NB4cells.Results: RT-PCR and Western blot showed that NE gene and proteinwere successfully expressed in NB4cells electroporated pCMV-HA-NE plasmid, and there did exist NLS-RARα expression. Using cellimmunofluorescence, confocal laser scanning, we found that NLS-RARαprotein was present in electroporated NB4cells and mostly localized incell nucleus.Conclusion: We have successfully electroporated pCMV-HA-NEplasmid in NB4cells and verified the existence of NLS-RARα protein withWestern blot, immunofluorescence, confocal laser scanning method.NLS-RARα was mainly localized in nucleus. PART Ⅱ Location Verification of NLS-RARα Protein in thesubcutaneously transplanted tumor of electroporated NB4cells in nudemiceObjective: Location verification of NLS-RARα protein in thesubcutaneously transplanted tumor of electroporated NB4cells in nudemice.Methods: We electroporated pCMV-HA-NE plasmid into NB4cells, and Western blot were used to verify the NE protein expression.Established the subcutaneously transplanted tumor models of electroporated cells in nude mice. Extracted the nucleoprotein oftransplanted tumor cells, using Western blot method to determine theexpression of NLS-RARAα in tumor cell nucleus. FITC-Annexin V/DAPIdouble staining immunofluorescence, confocal laser scanning method andimmunohistochemistry were used to examine NLS-RARAα proteinexpression and localization in transplanted tumor cells.Results: We have successfully electroporated pCMV-HA-NEplasmid into NB4cells, and established the subcutaneously transplantedtumor models of electroporated cells in nude mice. Verified the existenceof NLS-RARα protein with Western blot, immunofluorescence, confocallaser scanning method and immunohistochemistry. NLS-RARα was mainlylocalized in nucleus.Conclusion: These results provided a new way for further study inearly diagnosis and monitoring of acute promyelocytic myeloid leukemia. PARTⅢ Location Verification of NLS-RAR protein in APLpatients blood neutrophilsObjective: To verify the location of the NLS-RAR α protein in the neutrophile granulocyte ofAPL patients’peripheral blood.Methods: Western blot was used to identify the NE enzyme inneutrophile granulocyte of APL patients’ peripheral blood; neutrophilsnucleoprotein was prepared from APL patients’ peripheral blood.NLS-RARα protein was detected by Western blot;The expression andlocation of NLS-RARα protein in peripheral blood of patients with APLwere determined by FITC-Annexin V/DAPI double immunofluorescencestaining and FITC-Annexin V/PI double staining laser confocal microscopy.In our study, the expression and location of NLS-RARα protein in NB4cells electroporated pCMV-HA-NE plasmid as positive control.Results: NE enzyme protein were expressed in patients’ peripheralblood with APL. Verified the existence of NLS-RARα protein inneutrophile granulocyte ofAPL patients’peripheral blood withWesternblot,immunofluorescence, and confocal laser scanning method. NLS-RARα wasmainly localized in nucleus.Conclusion: NLS–RARα protein was successfully detected throughthree different methods in neutrophile granulocyte of APL patients’peripheral blood.