| Regulation of gene expression is a prominent component of a complex network. and Gene regulation is primarily achieved at the level of gene transcription.Different genes are being transcribed in different cell types and respond to various stimuli,such as hormones,growth factors,stress,injury,and inflammation,which control cell cycle progression,cellular proliferation, differentiation and cell mobility,and interactions with neighboring cells.The studies show that transcription factor pay a pivotal role in the gene transcription.The enormous advances in genetic engineering techniques over the last decade have made possible their identification and elucidation of their structure and the signaling pathways that modulate their function.NFκB was first identified as a B-cell nuclear factor and given its name on the basis of its ability to bind to an intronic enhancer of the immunoglobulin κ-light chain gene by Ranjan Sen in 1986. up to now, five mammalian NF-κB family members have been identified and cloned,and found in many other cells . NFκB play an important role in the inflammation,immune response,cell proliferation,cell apoptosis. After more than a decade of intensive studies,it is confirmed that NFκB a pivotal transcription factor governs the expression of early response genes involved in cell-to-cell interaction,intercellular communication,cell recruitment or transmigration,amplification or spreading of primary pathogenic signals,and initiation or acceleration of tumorigenesis.Because of its key role in the inflammtion and tumorigenesis, this transcription factor has great potential as targets for therapeutic. Objective: The aim ofcurrent research is To investigate the regulation of nuclear factor κB on tissue factor in fresh Acute promyelocytic leukemia cells ,and the modulating effect of Panax notoginseng saponins(PNS) on tissue factor through the expression of NFκB and IκB in NB4 cells. It is anti-coagulant that PNS down-regulates TFmRNA and tissue factor antigen level in NB4 cells,and influences differentiation induction, which will provide the basis for the development of new therapeutic strategy and approach for DIC.Method 1 cell isolation and culture The cells were obtained from the bone marrow of APL patiens and the peripheral blood of normal volunteers. APL cell line NB4 cell were cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS),100U/ml penicillin and 100μg/ml streptomycin in a fully humidified atmosphere of 5%CO2 at 37℃.The cells were seeded at a density of 5×105 cells/ml in 25cm2 culture flasks,and passaged every two days.Logarithmically growing cells with viability≥95% as assayed by trypan blue exclusion staining were used in the experiment.PNS dissolved in RPMI1640 free FBS to make a stock of 100mg/ml and solution in the experiment.2 The expression of TF and IκBαmRNA by RT-PCR Total cellular RNA was extracted from APL cells or NB4 cells by the TRIzol methods.M-MLV RTase was used for reverse transcriptional synthesis of cDNAs.TF primer pairs were5'- GAA GGA ACA ACA CTT TCC TA-3',and5'-GG GCT GTC TGT ACT CTT CCG GTT A-3'(213bp),amplification was conducted with 35 cycles of 30s at 94℃,30s at 60℃, and 45s at 72℃. IkBαprimer pairs were5'-tattctccctaccagctcac-3'and 5'- cactcctggctgttacatgtcac-3'amplification was conducted with 30 cycles of 40s at 94℃,40s at 58℃, and 1minutes at 72℃. PCR products were resolved by electrophoresis 90V 40minutes in 1.8% agarose gel with ethidium bromide staining,visualized under ultraviolet light,and quantified by an Alpha 　ImageTM 1220 & documentation Analysis System .3 The proliferation inhibitory effects of PNS on NB4 cells Cells in exponential growth were exposed to various concentrations of 96-well flat-bottomed in microplates.Six hours before the ending of culture, 20μl of MTT solution(5mg/ml)was added to the cells in each well.After 10minutes of centrifugation,the supernatants were discarded, the precipitate was dissolved in DMSO,and the amount of...
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