Investigation Of Neutropil Elastase In The Development Of Leukemia Cells

PART ⅠCONSTRUCTION OF RECOMBINANT LENTIVIRAL CARRYING HUMAN HNE GENE AND ITS SCREENING IN NB4 CELLSObjective: To construct a lentivirus vector that was inserted the NE gene.The acute promyelocytic leukemia cell line NB4 was infected with LV5-NE and the positive infected cells were screened using puromycin.Methods: The CDS of NE was obtained by PCR with p CMV-myc-NE as template. Verify the NE gene by enzyme digestion after connected to the shuttle vector p GLV10/U6/GFP/Puro. The correct plasmid and packaging plasmid(p Gag/Pol、p Rev、p VSV-G)were co-transfected HEK 293 cells to obtain recombinant lentivirus. Detect the titer of lentivirus.There were control group, NB4/LV5-NC group and NB4/LV5-NE group in this experiment.The infection efficiency of LV5-NE in NB4 cells was observed by GFP expression using fluorescence microscopy.The NB4 cells were screened with 1 μ g/m L puromycin after infected by lentivirus for 72 h.The expression of NE in NB4 cells was detected by reverse transcription-polymerase chain reaction(PCR), quantitative PCR; western-blot was used to further identify the expression of NE in NB4 cells.Results:NotⅠ and Bam HⅠ digestion and sequences test suggested that the LV5-NE recombinant vector expressing NE were successfully constructed,and the titer of lentivirus was 3ⅹ108. GFP expression observed by fluorescence microscopy implied the highly infection efficient of LV5-NE in NB4 cells. The pure NB4/LV5-NE and NB4/LV5-NC cells were used for subsequent experiments. RT-PCR and q RT-PCR results showed the m RNA of NE was expressed in the NB4/LV5-NE cells(P<0.05). The western-blot result showed NE was protein was expressed in the NB4/LV5-NE cells(P<0.05). The percentage of GFP-positive cells was about 100%, showing that all cells had been infected after screening with puromycin.Conclusion: We successfully constructed LV5-NE lentivirus expression vector and successfully infect NB4 cells. The recombinant lentivirus of LV5-NE was essential for exploring the possible mechanism in NB4 cells.PART Ⅱ THE EFFECTS OF NEUTROPHIL ELASTASE ON PROLIFERATION, APOPTOSIS AND PI3K/AKT SIGNAL PATHWAY IN NB4 CELLObjective: To investigate the effect of NE on proliferation, apoptosis and PI3K/Akt signal pathway in NB4 cells.Methods: There were control group, NB4/LV5-NC group and NB4/LV5-NE group in this experiment. The NB4 cells were screened with 1 μ g/m L puromycinafter infected by lentivirus for 72 h.The q RT-PCR was used to detect the m RNA of NE in NB4 cells;western-blot was used to further confirm the protein of NE expression; cck-8 was used to detectthe biological effects of NE in NB4 cells.Flow cytometry was used to detect cell cycle and apoptosis;q RT-PCR was used to detect the m RNA of CCD1,FOX1,m TOR,RICTOR and PTEN in NB4 cells; western-blot was used to detect p Akt, Akt, apopyosia-related genes Bax, bcl-2 expression.Results: The CCK-8 assay showed that over-expression of NE could promote the proliferation of NB4 cells when compared with that of the control cells(P<0.05). The flow cytometry assay showed that the percentage of NB4/LV5-NE cells in S phase and G2 phase were significantly higher than that of NB4 cells or NB4/LV5-NC cells and the percentage of apoptotic NB4/LV5-NE cells was significantly lower than that of NB4 cells or NB4/LV5-NC cells(P<0.05). The q RT-PCR assay showed increased expression of gene CCD1 and FOX1 in NB4/LV5-NE cells when compared with NB4 cells and NB4/LV5-NC cells while m TOR, RICTOR and PTEN were decreased(P<0.05). The western blot assay showed increased expression of phosphorylated(activated) Akt and Bcl2 in NB4/LV5-NE cells than that in the NB4 cells or NB4/LV5-NC cells(P<0.05).Conclusion: Over-expression of NE promoted the proliferation and inhibited the apoptosis of NB4 cell. NE may promote NB4 cell proliferation by activation of the PI3K/Akt pathway.Part III THE EFFECT OF DOWN-REGULATION NE ON PI3K/AKT SIGNAL PATHWAY IN U937 CELLObjective: To explore the effect of down-regulation NE on the PI3K/Akt pathway in U937 cells.Methods: si RNA-NE was transfected in U937 cells with the help of lipofectamine 2000; fluorescence microscopy was used to identify the transfection efficiency;q RT-PCR was used to detect the m RNA of NE in U937 cells;western-blot was used to detect the expression of NE in U937 cells; q RT-PCR was used to detect the m RNA of CCD1, FOX1, m TOR, RICTOR and PTEN in U937 cells; western-blot was used to detect p Akt, Akt, apopyosia-related genes Bax, bcl-2 expression.Results: Fluorescence microscope showed transfection efficiency of si RNA-HNE was more than 95%; western-blot assay showed the amount of NE was significantly reduced;q RT-PCR assay showed decreased expression of gene CCD1 and FOX1 in U937/NE-sh RNA cells when compared with U937 cells or U937/sh RNA-NC cells while m TOR, RICTOR and PTEN were increased(P<0.05);the western blot assay showed that the expression of phosphorylated(activated) Akt and Bcl2 in U937/NE-sh RNA cells were lower than that in the U937 cells or U937/sh RNA-NC cells(P<0.05).Conclusion: si RNA-NE successfully reduced the expression levels of NE in U937 cells,then genes and proteins of PI3K/Akt were changed.This may suggested NE may promote U937 cell proliferation by activation of the PI3K/Akt pathway.