Study On The Anti-leukemia Mechanism Of Shikonin

ObjectivesThis experiment is an in vitro study of effects of shikonin on leukemia K562 cell and leukemia patients' marrow primary mononuclear cell in terms of proliferation and apoptosis. It makes a preliminary study of the anti-leukemia mechanism of action of shikonin, explores its optimal action concentration and action time in anti-leukemia in vitro, thus provides laboratory basis for using Shikonin in clinic treatment of leukemia.Materials and Experiment Methods1? Using shikonin with different concentration(0.5?1?2?4 ?mol/L)to act on K562 cell and leukemia primary cell and adopting trypan blue excluding to count number of living cells and draw the cell growth curve;2?MTT test is used to observe the proliferation inhibition effects of different concentration(0.5?1?2?4 ?mol/L)shikonin on K562 cell and primary marrow mononuclear cell,and draw the inhibition rate curve;3?Using DAPI staining method to observe its actions on cell nucleus with different concentration(0.5?1?2?4 ?mol/L)under the fluorescence microscope;4?Flow cytometry is used to test the apoptosis rate when shikonin with different concentration(0.5?1?2?4 ?mol/L)acts on K562 cell,and also the change of Fas/Fas L;5?Using western blot method to test cell apoptosis protein expression and related protein expression of signal pathway and preliminarily explaining the mechanism of action that how shikonin induces apoptosis of K562 cell and leukemia patients' primary marrow mononuclear cell.Results1? Trypan blue excluding test display: shikonin with different concentration(0.5?1?2?4 ?mol/L) can suppress proliferation within 12?24?36?48?72 hours, and its intensity depends on time and concentration.2?DAPI :With the increasing of concentration and extending of time K562 cellshows apoptosis of various degrees that from pyknosis, edge accumulation and gradually to nuclear fragment. This shows that shikonin can induce K562 cell apoptosis and this effect has dependence on time and concentration.3?MTT test finds out that all the shikonin with different concentration can inhibit K562 cell proliferation and the proliferation curve has dependence on time and concentration. The IC 50 concentration is about 1umol/L.4?Flow cytometry to detect apoptosis rate: results show sinistral shikonin in a certain time(12 h, 24 h) and the concentration range(0.5, 1, 2, 4 ?mol/L) can induce human leukemia K562 cells apoptosis, role along with the increase of drug concentration and action time has increased trend, and has dependence on time and concentration.5?Flow cytometry to detect Fas/FasL expression: flow cytometry instrument testing different sinistral shikonin concentration(0.5, 1, 2,4 ?mol/L) to act on human leukemia K562 cells for 24 h, and flow cytometry test Fas/FasL, the result shows that sinistral shikonin can rise Fas/FasL expression, and its action has the concentration dependence?6?Using western blot method to test the apoptosis protein expression:The results show that after being affected by shikonin,bax protein expression is up-regulated, BCL-2protein expression is down-regulated, caspase-3?caspase-8?PARP protein is activated and sheared, Activating Mitogen-activated Protein Kinese(MAPK) and inducing apoptosis through MAPK signal pathway,P-38?JNK pathway is phosphorylated. its action has has dependence on time and concentration.Conclusion1?Shikonin can inhibit human leukemia K562 cells and leukemia patients primary bone marrow mononuclear cell proliferation and induce its apoptosis,this action has dependence on time and concentration2?The mechanism of action of shikonin induced apoptosis apply to up-regulation bax,reduce bcl-2,up-regulation Fas/FasL,Activating caspase-3?caspase-8?PARP,activating Mitogen-activated Protein Kinese(MAPK) and phosphorylation P-38?JNK pathway?...