Reserch Of Effects On Proliferation And Apotosis Of NB4 Leukemia Cell By PML (ΔNLS) And Lithium Chloride

PART I CONSTRUCTION OF RECOMBINANT LENTIVIRUS LV5-PML(ΔNLS) And ITS EFFECTS ON APOTOSIS OF NB4 CELLSObjective: To study the effects of recombinant lentivirus LV5-PML(ΔNLS) on apoptosis of NB4 cellsMethods: PML(?NLS) gene was amplified by PCR using plasmid p CMV-HA-PML(?NLS) as a template and cloned into the lentivirus vector LV5-EF1a-GFP/puro.The recombinant lentiviral vector,p Helper 1.0 and p Helper 2.0 were co-transfected into 293 T cells,packaging virus. Then NB4 cells were infected by lentivirus LV5-PML(ΔNLS). PML(?NLS) m RNA transcription and protein expression in NB4 cells were determined by RT-PCR and Western blot respectively, the expression of Akt,p-Akt(ser473), S6 and p-S6(ser235/236) proteins in NB4 cells were analyzed by Western blot. Cell proliferation was detected by CCK-8. NB4 cells were treated with 100 nmol/L Rapamycin for 24 h, then cell apotosis and cycle were detected by FCM.Results: Recombinant lentivirus LV5-PML(?NLS) was successfully constructed. The infection efficiency in NB4 cells reached 82.74%,the PML(?NLS) gene in LV5-PML(?NLS) was expressed stably in NB4 cells; the expression of p-Akt(ser473) and p-S6(ser235/236) proteins were increased in NB4 cells infected with LV5-PML(?NLS). The proliferation of NB4 cells infected by LV5-PML(ΔNLS) was significantly enhanced as compared to the untransfeced and empty viral groups. The phase of G1 of infected NB4 cells was increased and the phase of S of NB4 cells was decreased by FCM assay.Conclusion: Recombinant lentivirus LV5-PML(?NLS) was constructed successfully. The Over-expression of PML(ΔNLS) could promote the proliferation of NB4 cells by acting the Akt signaling pathway and affect the level of p-S6 protein.PART II STUDY OF EFFECT OF LITHIUM CHLORIDE ON PROLIFERATION AND APOTOSIS OF NB4 CELLSObjective: To study the effect of lithium chloride on apotosis of NB4 cells.Methods: NB4 cells were cultured with 0-40 m M Li Cl for 24 h, Then the cytotoxic effect was detected by CCK-8 array, the expression of Akt1 was detected by Western blot. NB4 cells were treated with 20 m M Li Cl for 24 h, after treatment cell apotosis and cycle were detected by FCM assay, the expressions of b-catenin, p-GSK-3b, GSK-3b and c-Myc by Western blot.Results: The proliferation of NB4 cells was inhibited and the expression of Akt1 was decreased by Li Cl in a dose-dependent manner. 20 mmol/L concentration of Li Cl promoted apotosis and induced cell cycle arrested at G2/M phase in NB4 cells, the expressions of Akt1, c-Myc were significantly decreased and the expressions of b-catenin and p-GSK-3b were increased.Conclusion: Li Cl could promote apotosis in NB4 cells by inhibite GSK-3b. In addition, it could affect the expression of Akt1, b-catenin, p-GSK-3b, and c-Myc.