Effect Of Neutrophil Elastase Inhibitors On The Proliferation And Apoptosis Of U937 Cells

PART1 EFFECT OF NEUTROPHIL ELASTASE INHIBITORS ON THE PROLIFERATION AND APOPTOSIS OF U937 CELLSObjective: To study and compare the effect of neutrophil elastase inhibitors (GW311616A and sivelestat) on the proliferation and apoptosis of U937 cells.Methods: The experiment object-U937 cells were divided to 3 groups (control group, GW311616A group and sivelestat group). The proliferation and apoptosis of U937 cells was detected with MTT assay, transmission electron microscope, AnnexinⅤ-FITC/PI staining and flow cytometry. And the expression of NE in U937 cells was observed in indirect immunofluorescence, the variation of content and activity of NE in U937 cells was measured through ELISA assay and colorimetric method.Results: The two inhibitors could inhibit the proliferation of U937 cells in a dose dependent manner. But the inhibition of GW311616A was higher than sivelestat, it was statistical significance (P<0.01). The result of electron microscope showed the two inhibitors could cause the apoptosis of U937 cells. U937 cells could be induced to undergo apoptosis by the two inhibitors, but the effect of GW311616A on the apoptosis of U937 cells was significantly higher than that of sivelestat (P<0.01).The result of flow cytometry indicated that the cell cycle was blocked in S and G2/M phase, but mainly in G2/M phase, by GW311616A. Sivelestat could arrest U937 cell cycle in S phase. The decrease of fluorescence intensity was observed after U937 cells had been treated with 150μmol/L GW311616A for 48h. However, the fluorescence intensity of sivelestat group was obviously attenuated. And the two inhibitors could reduce the content and activity of NE in U937 cells, but the effect of GW311616A was significantly higher than that of sivelestat (P<0.01).Conclusion: GW311616A and sivelestat could inhibit and cause the proliferation and apoptosis of U937 cells. Nevertheless, GW311616A was more effective and harmful to cells, compared with sivelestat. PART2 INTERACTION BETWEEN NLS-RARαAND UBIQUILIN1Objective: To identify the interaction between NLS-RARαand Ubiquilin 1(UBQLN1).Methods: The recombination expression plasmids pGBKT7-NLS-RAR and pACT2-UBQLN1, which expressed bait-protein NLS-RARαand target protein UBQLN1 respectively, were cotransformaed into yeast AH109 and the interaction of the expression plasmids in the living cells was investigated by yeast two-hybrid assay. HA-tagged fusion protein (pCMV-HA-NLS-RARα) expression vector and Myc-tagged fusion protein (pCMV-Myc-JTV1) expression vector were respectively constructed, identified, and cotransfected into HEK293. The interaction was detected by co-immunoprecipitation.Results: Blue clones were found on SD/-Ade/-His/-Leu/-Trp/X-α-gal plate. Double restriction enzyme digestion showed that pCMV-HA-NLS-RARαand pCMV-Myc-JTV1 were successfully constructed. Then HA-NLS-RAR protein was immunoprecipitated by anti-HA polyclonal antibody and the expression of Myc-UBQLN1 was tested by western blot with anti-Myc monoclonal antibody from immunoprecipitated complex. Conclusion: The interaction between NLS-RARαand UBQLN1 was existed by yeast two-hybrid assay and co-immunoprecipitation.