Study Of The Signal Transduction Mechanism On The Effect Of Angelica Polysaccharide On Inhibition Of Proliferation And Induced Differentiation Towards Erythroid Cell In K562 Cells

Leukemia is a malignant hematopoietic tumor which threatens the health of humanity.Until now,traditional chemotherapy and radiotherapy are still the main therapies,but the therapy have many side effects and the ratio of relapse is very high.What's more,it greatly damages the immunity and hematopoiesis.In recent years,finding effective ingredients has become the hope for the therapy of leukemia.More and more scientists think that the Chinese traditional medicines can play an important role in the therapy for it's two sidea affection on body.Finding natural effective ingredients extracted from Chinese traditional medicine which has both the effect of promoting normal hematopoiesis and the effect of induce the leukemia cell differentiate towards normal state become the new hope of leukemia therapy.Objective: Our group have certified that APS not only can intensify normal hematopoiesis,but also can inhibit the proliferation of leukemia cells,while inducing them differentiate towards maturity,and the mechanism is not clear.During hemopoiesis,Epo combined with EpoR is very important. JAK2 and STAT5 play the key role in the pathway of signal transduction which mediated by Epo.To investigate the effect of Angelica polysaccharide on inhibition of proliferation and induced erythrocytic differentiation in K562 cells. To approach the effect of Angelica polysaccharide (APS) on JAK2/STAT5 signal transduction of K562 cells and to explain the molecule mechanism for the regulation of APS on hematopoietic cell proliferation and differentiation.Methods: 1,Techniques of cell culture were used in this experiment.The effect of APS on proliferation of K562 was examined by MTT,trypan blue staining and flow cytometry; 2,The change of cell form was observed by Wright's stain;3,The effect of APS induced erythrocytic differentiation of K562 was detected by cytochemistry and spectrophotometric method.;4,To observe the distribution of JAK2 and STAT5 in K562 treated with APS at different conditions( different concentrations of APS and different treated times by immunocytochemistry and laser confocal microscopy;5,To detect the expression of JAK2 and STAT5 in nucleus and cytoplasm of K562 cells treated with APS at different times by Western blotting ; 6,To detect the expression of JAK2 and STAT5 in nucleus and cytoplasm of K562 cells treated with APS combined with Epo(5×103 IU/L)by Western blotting; 7,To detect the activity of JAK2 and STAT5 in nucleus and cytoplasm of K562 cells treated with APS combined with Epo(5×103 IU/L)by immuno-precipitation;8,To observe the distribution of JAK2 and STAT5 by ICC and fluorescence microscope. Results: 1,APS can significantly inhibit the proliferation of K562 ex vivo,and the inhibitory extent is paralleled to the concentration and the acting time of APS;2,APS may prevent K562cell of the silent phase from entering the active proliferation phase;3,After the effection of APS the volume and diameter of K562cell,the ratio of nucleus / cytoplasm are decreased,while the chromatin in the nucleus increased;4,APS promoted the expression of Hb.;5,The expression of STAT5 in cytoplasm of APS treated K562 cells is obviously less than that of group negative control for 12h, but the expression of STAT5 in nucleus of APS treated K562 cell is markedly stronger. It is not obviously differences between the APS treated cell and negative control cell for 72h;6,The results showed that the expression of STAT5 in nucleus of APS treated cell is stronger than that of group negative control cell at 12h,24h and 48h, but the expression in cytoplasm was weaker.The expression of STAT5 in nucleus of APS treated cell is weaker than that of group negative control cell at 72h.STAT5 in nucleus in the cell treated hydrea at 24h increased obviously;7,The expression of JAK2 is not difference between the APS treated cell and negative control ;8,The activity of JAK2 in APS group is stronger than that in control group effected with Epo;9,Both the activity and the expressin of STAT5 in APS group is stronger than that in control group effected with Epo.Conclusion: APS can significantly inhibit the proliferation and induce the differentiation towards erythroid cell of K562 ex vivo. STAT5 is transfered from cytoplasm into nucleus when APS treated cell for 48 hours, but the nuclear translocation is inhibited for 72h. The expression of JAK2 is not obviously influenced by APS, which pointed out that APS regulate the hematopoiesis is relevant to JAK2/STAT5 signal transduction.We can drow an conclusion that APS not only can intensify normal hematopoiesis,but also can inhibit the proliferation of leukemia cells,while inducing them differentiate towards maturity.