Left-handed Alkannin Research On The Effect Of Chronic Myelogenous Leukemia

Experimental objective:Preliminary study of sinistral alkannin primitive cell leukemia,chronic granulocyte leukemia K562 cells as well as the role of mononuclear cells normally,to expand sinistral alkannin in the treatment of chronic myelogenous leukemia in terms of potential application value.Experimental materials and experimental methods:Left-handed alkannin buy in Shanghai wan jiang biological technology co.,LTD.Select 1μmol/Lrespectively,2μmol/L、3μmol/L in leukemia K562 cells(by shaanxi province people’s hospital blood medical laboratory subculture)in a traditional way,in 0h、12h、24h 、 48 h,inverted microscope and fluorescence microscope(DAPI staining) at different drug concentrations cell morphological changes; trypan blue staining and determined by MTT colorimetry todetect proliferation inhibition rate of different drugs concentration effect; Flow cytometry dection of different drugs concentration cell apoptosis rate.Also collected five clinincal first diagnosis of chronic myelogenous leukemia patients peripheral blood,peripheral blood and in 3 normal after separation of solid extracted through Ficoll density gradient centrifugation separation from a single nuclear fine. Select 2μmol/L 、 4μmol/L sinistral shikonin,primitive cells function in chronic myelogenous leukemia and normal mononuclear cells,selection of main testing time points of 0h、24h、48h,trypan blue staining testing different drug concentrations from the single nuclear cell proliferation inhibition rate;Using the method of rael-time quantitative PCR testing different drug concentrations of primitive mononuclear cells in chronic myelocytic leukemia P210 BCR/ABL fusion gene expression rate.Experimental results:1.(DAPI staining) under inverted microscope and fluorescence microscope to observe sinistral alkannin leukemia K562 cells after 24 h of morphological changes:the different concentrations of sinistral alkannin(1μmol/L 、 2μmol/L 、3μmol/L)on K562 cells after 24 h,form a complete control cells,refraction good;Drug concentration 1μmol/L visible minority cell swelling,form become irregular;Drug concentration 2μmol/L cells form is more irregular,there is a small amount of apoptotic bodies;3μmol/L group appear a large number of apoptotic bodies and debris;2.The trypan blue staining, determined by MTT colorimetry to detect sinistral alkannin on leukemia K562 cell proliferation inhibition: different concentrations of sinistral alkannin(1μmol/L、2μmol/L、3μmol/L)in 12h、24h、48h can inhibit the proliferation of K562 cells,and its inhibition has the time and the concentration dependence;3.Flow cytometry detection sinistral radix arnebiae seu lithospermi effect on apoptosisof leukemia K562 cells:different concentration sinistral alkannin(1μmol/L、2μmol/L、3μmol/L)cells K562 cells for 24 h after the analysis of early and late apoptosis rate of K562 cells group,respectively(4.85±0.34) %( 1μmol/L),(10.53±0.72)%( 2μmol/L),(18.71±1.34)%(3uoml/L),and the control group(3.95±0.46)%. Compare different sinistral shikonin concentration process cells measured after each cell apoptosis rate:the experimental group(1μmol/L)apoptosis rate difference compared with control group no significant statistical significance(P>0.05);The experimental group(2μmol/L、3μmol/L)and the control group and cell apoptosis rate compared to the differences between the experimental group were statistically significant(P<0.05);4.Determination of trypan blue from dying sinistral alkannin primitive cells in chronic myelocytic leukemia and normal mononuclear cell function: the influence of different concentration of sinistral alkannin(2μmol/L、4μmol/L),respectively,primitive cells function in chronic myelogenous leukemia and normal mononuclear cells in different time(0h、12h、24h、48h)after living cell count with trypan blue from dying and scaling inhibition rate, found umol sinistral shikonin concentration level can obviously inhibit the generation of chronic myeloid leukemia cell proliferation(P<0.05),and its inhibition concentration and time dependence;And the same concentration of sinistral alkannin to not significantly affect the normal mononuclear cell proliferation(P>0.05);5.Real-time quantitative PCR detection P210 expression rate:sinistral shikonin concentration(2μmol/L、4μmol/L)role in primitive cells of chronic myelogenous leukemia after 24 h the real-time quantitative PCR detection results showed μmol/L concentration level sinistral alkannin could significantly cut the primitive cells of chronic myelogenous leukemia BCR/ABL fusion gene expression of P210(P<0.05).Experimental conclusion:Left-handed alkannin proliferation of leukemia K562 cells have obvious inhibitory effect,the inhibition of strength in concentration and time dependence;Sinistral alkannin in on normal mononuclear cell proliferation,in the absence of any obvious effect inhibits the proliferation of chronic myelogenous leukemia primary cell and induce its apoptosis,and the BCR/ABL fusion gene expression of P210 has obvious effect.