Investigation Of Neutropil Elastase In The Development Of Leukemia

CONSTRUCTION OF RECOMBINANT ADENOVIRUS CARRYINGHUMAN HNE GENE AND ITS EFFECTS ONK562CELLSObjective: to observe the biological effects of NE on K562cellsproliferation and apoptosis,AdEasy system was used to constructrecombinant adenovirus which bring HNE gene and then over expressedNE in CML cell line K562.Methods: The Ad-NE recombinant vector expressing NE wereconstructed, then transfected HEK293cells to obtain recombinantadenovirus. when get the first recombinant adenovirus, expanded in HEK293cells. The infection efficiency of AdE-NE in k562cells was observedby GFP expression using fluorescence microscopy; western-blot was usedto further identified the expression of NE in k562cells. to research thebiological of NE in k562cells,CCK-8method was used to obtain theproliferation situation, Flow Cytometry was used to analysis cell cycles andapoptosis. The expression of CyclinB1、Bax and Bcl-2was analysised by western-blot.Results: HindⅢ and EcoR V digestion and sequences test suggestedthat the Ad-HNE recombinant vector expressing NE were successfullyconstructed;GFP expression observed by fluorescence microscopy impliedthe highly infection efficient of Ad-NE on K562cells. western-blot resultsshowed NE was up regulated in K562cells. Cells cycle analysis showedK562cells were blocked in G0/G1phase. Apoptosis results showed that theapoptosis ratio of K562was highly increased.Conclusion: We successfully constructed Ad-NE adenovirusexpression vector. NE can promote leukemia cell proliferation, inhibition ofapoptosis In CML cell line K562,. NE may play a catalytic role in thedevelopment of leukemia. THE Effect OF DOWN-REGULATION NE ON U937CELLPROLIFERATION ANDAPOPTOSISObjective: Down-regulate NE in U937cells and investigate theproliferation and apoptosis of U937cells. Methods: siRNA-NE was transfected in U937cells with the help oflipofectamine2000; fluorescence microscopy was used to identify thetransfection efficiency;western-blot to further confirm the down-regulationof NE expression; flow cytometry was used to detect cell cycle andapoptosis; western-blot evaluated apopyosia-related genes Bax, bcl-2expression.Results: Fluorescence microscope showed transfection efficiency ofsiRNA-HNE was more than95%; western-blot results showed NE contentwas significantly reduced; flow cytometry showed that cell cycle wasarrest in G0/G1phase and increased apoptosis; western-blot showedapoptotic-regulated protein Bax was up-regulated, inhibition of apoptosisprotein Bcl-2was down-regulared.Conclusion: siRNA-NE successfully reduced the expression levels ofNE in U937cells, and promote apoptosis by down-regulated Bcl-2andraised Bax. GW311616A ON PROLIFERATION AND APOPTOSIS OF HL-60CELLSobjective: To study the effect of GW311616A on the proliferation andapoptosis of HL-60cells.Methods: Inhibitory effect of GW311616A on the proliferation ofHL-60cells was assayed by CCK-8assay. The morphologic changes ofHL-60cells were detected by microscope and apoptosis was observed byAnnexinⅤ-FITC/PI staining. The changes of cell cycle and apoptosis weredetected by flow cytometry. The variation of content and activity of NE inHL-60cells was measured through colorimetric method.Results: CCK-8showed GW311616A could inhibit the proliferationof U937cells in a dose dependent manner. Typical apoptosis morphologicalchanges of HL-60was observed through microscope. AnnexinⅤ-FITC/PIstaining showed that HL-60cells could be induced to undergo apoptosis byGW311616A, the apoptosis ratio was36.44%, cell cycle was mainlyblocked in G0/G1phase.Conclusion: GW311616A could inhibit the proliferation and causeapoptosis of HL-60cells.