| ObjectivesTo detect the differentially expressed proteins of mononuclear cells in bone marrowfrom AML patinents by proteome technique and to search the unknown proteins indatabank，Further,to discuss the relation between these proteins and multidrugresistance,then provide the basis of multidrug resistance for early diagnose.Methods1.Patinents and sample Sample were derived from bone marrow aspirates of16patients age13–67years(median,37.4years) with acute myeloid leukemia.Thediagnosis was made according to the WHO classification system on the basis ofmorphology, clinical manifestation,immunophenotyping and genetics.The patientswere divided into drug resistance group and chemo-responsive group base on theresponse.2. Two-dimensional electrophoresis Cells were disrupted by Cell lysis buffer andprotein sample was extracted by freeze thawing and subpackaged, then quantified.Application of IPGphor3isoelectrofocusing system and Ettan DALTsix Largevertical electrophoresis system to carry out two-dimensional electrophoresis,Imagemaster2D platinum software analyzed gel and found differentially expressedprotein spots.3. Mass spectrographic analysis Cut down the protein spots with Significantdifferences and enzymatic lysis, then utilize mass spectrographic to analysis thepeptide.4. Searching the Protein database Put the peptide fragment into database to ensurethe proteins after obtain the accurate peptide mass fingerprint of protein points.ResultsRepeat three times each sample with two-dimensional electrophoresis and the resultgot a better repeatability. ImageMasster2-D detect638±39protein spots ofmultidrug resistance group and704±34protein spots of Chemo-responsivegroup.There are10high expressed protein spots in multidrug resistance group and19 high expressed protein spots in Chemo-responsive group after ImageMasster2-Dsoftware analyzed. Cut5spots with significant differences in each group.A total of10spots with significant differences were analyzed by mass spectrometry,3protein spot’s score below60and been considered as unlikelihood. There had7spots been analyzed by mass spectrometry,8and25、36and37are the same spot, sothere had5spots been identified, as follow: Filamin-A、Actin、Peroxiredoxin-6、Fibrinogen gamma chain、60kDa heat shock protein。ConclusionThere are significant difference between multidrug resistance group andChemo-responsive group, multidrug resistance group high expressedPeroxiredoxin-6and60kDa heat shock protein with a poorresponse;Chemo-responsive group high expressed Filamin-A、 β-Actin andFibrinogen gamma chain with a better response.Those proteins may associate withthat leukemia cells response to the chemotherapeutic,then introduce multidrugresistance, have a instructions function to predict drug resistance. |
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