| There are thousands of years that Angelica is an important Chinese crude drug to nourish blood and promote blood flow.In past years,we domestrated that:1.Angelica polysaccharide(APS) is one of the main chemical compositions in.Angelica.2.APS cooperated with exogenous hematopoietic growth factors(Epo,GM-CSF,IL-3,et al) could obviously promote the proliferation and differentiation of BFU-E,CFU-E,CFU-GM and CFU-MK in health and anaemic mice.3.APS also could promote the proliferation and differentiation of BFU-E,CFU-E,CFU-GM and CFU-Mix in the human being,APS could promote the role of hematopoietic factor.APS has both the effect of promoting normal hematopoiesis and the effect of inducing the leukemia cell's differentiation towards normal state,APS has prompted a two-way control,but the specific molecular mechanisms is unclear.In recent years,studies have shown that JAKs/STATs mediated by hematopoietic growth factor(janus activated kinase/signal transducer and activator of transcription) signaling pathway is the key ways of many cytokines that regulate cell proliferation,differentiation and apoptosis.STAT5 is an important member of the family STATs,JAK2/STAT5 signal transduction pathway plays an important role in normal and leukemic hematopoietic system mediated by Epo,STAT5 as the Epo-Epo-R downstream elements of the original stem cells to maintain and extend the increase is necessary.abnormal activation of JAK/STATs pathway is closely related to the occurrence of leukemia,in leukemia cell lines and primary leukemia cells, there is a high level of STAT3 activation that promote tumor cell proliferation,inhibition of apoptosis in the normal process,more and more research indicate that STAT3 signaling pathway has proved to be a wide range of effective intervention in the treatment of tumor target molecule, STAT3 targeted therapy cancer treatment has become one of the hot area of research.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.In this research experimental subject are K562 cells and mononuclear cells that derived of human Umbilical cord blood,there are two parts in this research:1.Effects of angelica polysaccharide on expression of JAK2/STAT3 signal transduction molecule in K562 leukemia cells.2.Effect of Angelica polysaccharides co-erythropoietin on JAK2/STAT5 signal transduction pathway in hematopoietic stem/progenitor cells.Our research mainly study the two-way mediated mechanism of APS that promote haematopoiesis,Inhibite leukemia cell,proliferate and induce cell differentiation.This finding will provide theoretical and experimental evidence for exploiting and utilizing Angelica.Part 1:Effects of Angelica polysaccharide on expression and activiton of JAK2,STAT3 in K562 leukemia cellsObjective:To approach the influence of angelica polysaccharide(APS) on expression and activity of JAK2 and STAT3 in K562 cells,and reveal the molecule mechanism of the regulation of APS on differentiation and apoptosis of K562-cells treated with APS.Methods:1.group experiments:Control group,normal cultivated;APS group were cultured with Angelica polysaccharide(200 mg/L);cells in each group were collected for following research after culturing for.12,24,36,48,72 and 96h.2.To observe the expression of JAK2 and STAT3 in K562 cells induced by APS(200mg/L) at different times through ICC and laser confocal scanning microscope;3.To observe the expression of STAT3 in K562 cells induced by APS (200mg/L) at different times through laser confocal scanning microscope; 4.JAK2,Phospho-STAT3 and STAT3 in nucleus and cytoplasm were measured by western blotting in each group at different times.Results:1.The expression of STAT3 in nucleus of APS group was obviously less than that of control group for 12 h,but the expression in cytoplasm was opposite;2.There was no clear change of STAT3 protein in cytoplasm between APS group and control group for 72 h,expression of STAT3 protein in nucleus was down after treatment with APS for 72 hours.3.The results of western blotting showed that expression of STAT3 in cytoplasm was increased in APS treated cells at the time of 12h,24h,36 h,48h,4.compared with control group at different times,STAT3 protein in nucleus and phosphor-STAT3 were rapidly down in K562 cells induced by APS;Whereas the expression level of JAK2 remains unchanged.Conclusion:Expression of JAK2/STAT3 signal transduction molecule plays an important role in the effect of APS on proliferation,differentiation and apoptosis.Part 2:The effect of APS cooperated with Epo on activition of JAK2/STAT5 signal transdution pathways in umbilical cord blood hematopoietic cellsObjective:To investgate the signal transduction and regulation in erythropoiesis by Angelica polysaccharides(APS) to clarify the mechanism for APS promoting hematopoiesis.Methods:1.To isolate mononuclear cells from foetus umbilic cord blood(Mononuclear cells,MNCs),After MNCs were incubated in the presence of APS group(APS 200μg/ml) and control group for 24 h,the cells were stimulated with Epo(5 IU/ml) for 0,2,5,30 min;2.STATS was measured by ICC and laser confocal scanning microscope, JAK2,STAT5 in nucleus and cytoplasm were measured by western-blotting-ECL in each group at different groups.Results:1.Angelica polysaccharide cooperated Epo have significant impact on the expression of STAT5,expression of STAT5 have significant difference at 4 time points in APS group and the control group;2.JAK2,STAT5 expressed in cytoplasm and nuclear of APS group increased significantly than that of control group,and they expressed the strongest at 5 min.Conclusion:JAK2,STAT5 signal transduction molecule plays an important role in the effect of APS cooperated with Epo on promoting hematopoiesis.To sum up,Angelica polysaccharide can inhibit proliferation of K562 leukemia cells as well as the promotion of normal blood cells and their proliferation and differentiation,JAK/STATs signal transduction pathway is closely related to the impact.The further study of the bi-directional control mechanism of Angelica polysaccharide is important for the development of targeting therapy of leukemia drugs and stem cell mobilization induced by the natural agent to provide experimental basis.
|
PDF Full Text Request