The Experiment Research Of IDO Protein Expression Of Leukemia Cells Cultured With Tα1

PartⅠ: Tα1reduce the IDO protein expression of leukemia cell whichinduced by IFN-γObjective: Thymosinα1is a kind of small molecular polypeptidehormone which consists of28amino acids.Ta1can promote thesecretion of cells in the body and lymphocyte function, throughstimulates the lymphocyte mitogen to promote T lymphocyte mature,Ta1is a biological response modifiers. Indoleamine2,3-dioxygenase isa monomer protein which containing ferroprotoporphyrin.IDO is anextrahepatic enzyme that catalyzes the initial and ratelimiting step inthe degradation of tryptophan along the kynurenine pathway.Degradation of tryptophan can inhibit the proliferation of T cells andincrease the apoptosis, then leading to the immune tolerance.Inaddition,tryptophan toxic metabolites can directly suppresses thefunctions of T cells and induce T cell apoptosis.This experiment aims todiscuss the mechanism of Ta1downregulation indoleamine2,3-dioxygenase protein expression of leukemia cell which induced byIFN-γ.Methods:Use Western Blot method to detecte IDO proteinexpression of leukemic cells which induced by IFN/γ、Tα1/IFN-γ.1.Experimental group：①IFN-γ/Tα1inhibit IDO expression②IFN-γ/Tα1 reduce IDO express③Negative Contrast group,Each group givedifferent concentration and time treatment.2.Extracting cell protein, anduse Commassie Blue staining method to detect cells totalprotein.3.Western Blot test the protein expression of each group andwith ECL dyeing, multiple color fluorescence imaging camera.Results:1.Before add IFN-γ, IDOmRNA present a faint expression onthe map,IDO protein strip not seen clearly on map,After IFN-γ Induced,IDO protein expression obviously increased.2.After IFN-γ induced,then add Tα1,IDO protein strip significantly weakened with theincrease of Tα1’s concentration and processing time, When theconcentration of Tα1is0.2ug/ml, the inhibition rate of HL-60is39.92%, the inhibition rate of K562is20.59%;When the concentrationof Tα1is5ug/ml, the inhibition rate of HL-60is80.24%, the inhibitionrate of K562is61.76%.concentration groups:difference between HL-60groups is (F=4187.76P <0.001), K562is (F=59.497P <0.001),have significant statistical significance; Time groups: P <0.05,with a statistical significance.3.When IFN-γ/Tα1effect in HL-60orK562cells, IDO protein channel significantly weakened with theincrease of Tα1concentration and processing time,Indicates Tα1canweaken the inducement function of IFN-γ to IDO, expression reduced.statistical results:concentration groups:differences between HL-60groups is (F=117.7P<0.001), K562is (F=1382.67P<0.001),have a significant statistical significance.Conclusion:1.Usually,IDO express alittle in leukemia cell.2.IFN-γ can significantly raise IDO proteinexpression. After add IFN-γ, IDO express clearly enhance, tip IFN-γmay promote the leukemia cells immune tolerance, leading to a largenumber of tumor cell growth.3.Tα1reduce IDO protein expression withconcentration dependence and time dependence.4.Tα1inhibit IDOprotein expression.When IFN-γ/Tα1both act on HL-60or K562cells,IDO protein expression reduced,Hint Ta1can weaken the inducementfunction of IFN-γ to IDO. PartⅡ: The effect to the proliferation of T cellsafter reducingIDO+leukemia cell by Tα1Objective:Discuss the mechanism and inhibition to T cells after IDO+leukemia cell reduced by Tα1.Methods:1. IFN-γ induces K562orHL-60cells:IFN-γ(1000IU/ml) induced HL-60or K562cells72hours.2. Mix cultivate HL-60or K562cell and peripheral bloodlymphocytes:Trials are divided into①Hγ/kγ-irradiation MlR-PHAgroups;②Hγ/kγ-MlR-PHA+Tα1groups;③Hγ/kγ-MlR-PHA+1-MTgroups，Each groups were to drug induced72hours.3.Use MTT methodto detect T lymphocytes inhibition rate:completing the training afterreacting with MTT about4hours, add dimethyl sulfoxide to dissolve.4.Enzyme league immune detector570nm detect OD digital and analysethe data.Results:1.The PHA have no significant effect on the shape andnumbers of HL-60or K562cells.2. PHA can promote T lymphocyteproliferation and differentiation.Trough electron microscopyobservation,we can find that the numbers of lymphocyte increased, andincreased in size.3.Data analysis:The inhibitory effect to T lymphocytesof Hγ/Kγ-MlR-PHA groups is more apparent than H γ/kγ-MlR-PHA+Tα1group and H γ/kγ-MlR-PHA+1-MT group, and the rate of growthis lower,After three groups drugs induced respectively,whenT/HL-60=1:20（F=72.647, P <0.001）,with statistical significance; when T/HL-60=1:50（F=38.141,P <0.001）,with statistical significance.Conclusion:1.The PHA have no significant effect to Leukemia cells,the results show that the mixed cultivating have no influence on theresult of the experiment.2. PHA can promote T lymphocyteproliferation and differentiation,After induced by PHA,the T lymphoidcell quantity increased and the volume enlarged.3.Tα1down-regulatethe action of IDO on repressing T lymphoid cell. After PHA induced,Tlymphoid cell in great quantities proliferation,after add leukemia cell,the T lymphoid cell proliferation is repressed, but after adding Tα1and1-MT respectively, the T lymphoid cell inhibition rate step-down,Tipthat Tα1may as same as1-MT can reduce the inhibition to the Tlymphoid cells by the way of down adjust or repress leukemia cell IDOexpression.