The Changes Of Tissue Factor During The Process Of Differentiation Of K562Induced By Phorbol Esters

ObjectiveTissue factor(TF),also called coagulation factor III.TF not only involved in the pathophysiological process of adjustment and coagulation-related diseases,but also participate in the identification and regulation of intracellular and extracellular signals involved in signal transduction pathway.lt is reported in the literature:TF’s mRNA and protein level was elevated in human advanced tumors,including colorectal cancer,pancreatic cancer,leukemia, lymphoma and so on.However,the specific expresion of tissue factor and its specific regulatory mechanism is not clear.K562cells is isolated from patients with chronic myeloid leukemia(blastic phase),which have the characteristics of bone marrow hematopoietic stem cells.It not only can proliferation under normal culture conditions,but also can differentiation to erythroid,granulocyte and megakaryocytic in some special culture conditions.As a result,k562cells is an ideal model of leukemia cells.In the eighties of last century,it is discovered that phorbol ester(PMA) can induce differentiation of k562cells to megakaryocyte.At present,.whether the expression of tissue factor on the k562cell is still controversial and the changes of tissue factor during the process of differentiation of k562induced by phorbol esters is not clear.Therefore,this paper aims to observe whether tissue factor express on the k562cell surface and the changes of tissue factor during the process of differentiation of k562induced by phorbol esters.As a result,we can explore its significance in its clinical efficacy.Methods1.Human erythrocyte leukemic myeloid cell line k562were cultured.2.phorbol ester(PMA) induced Human erythrocyte leukemic myeloid cell line k562.3.After48h induction,a small amount of cell line were taked to Wright stain and obserced changes in the cell morphology4.CD33and CD41on the cell surface were detected through flow cytometry,which is used to indentify the direction of cell differentiation.5.mRNA levels and protein levels of tissue factor were respe-ctively detected by reverse transcription PCR(RT-PCR) and Western Blot.Results1.Wright stain:k562cells,under phorbol ester(PMA) lOng/ml concentration, emerged the morphological characteristics of the megakaryocytic cell:cell body increased;cell morphology was round or oval and its margin was irregular;chromatin was purple、coarse granular、 closely arranged;nulceous is not very clear.2.The expression of CD33and CD41on the original k562cell respectively were80.49±0.7%,0.78±1.28%,while after induced for48h,17.92±2.9%,49.82±6.1%.3.Tissue factor’s mRNA and protein expression levels on the original k562cells was positive.4.RT-PCR:TF expression at the RNA level on the k562cells induced by phorbol ester began to decline on the six days,while seven days of expression is lower compared to six days.5.Western Blot:TF expression at the protein level on the k562cells induced by phorbol ester began to decline on the seven days.Conclutionsk562cells have the expression of tissue factor.what’s more,k562cells,under phorbol ester(PMA) lOng/ml concentration, were successful induced to megakaryocytic cell. mRNA levels and protein levels of tissue factor on the surface of k562cells induced by phorbol ester(PMA) reduced.