| Purpose:This experiment is based on the theory of "Qi is the blood and blood is the mother of Qi",Selection of classic Chinese medicine supplementing prescription of Danggui Buxue Decoction effective components of Angelica and astragalus polysaccharide for drug intervention, with Angelica polysaccharide, astragalus polysaccharide and combined medication as the experimental group with recombinant human erythropoietin as positive drug control group, inhibition of murine hematopoietic stem cells as the experimental object by intraperitoneal injection cyclophosphamide induced bone marrow,To observe the effects of Angelica sinensis polysaccharide, astragalus polysaccharide and combination therapy on hematopoiesis in mice with bone marrow suppression,Based on the JAK2-STAT5 signaling pathway and the factors that affect the hematopoietic cells, the possible mechanism of Angelica sinensis polysaccharide, astragalus polysaccharide and the combination of drugs were investigated.Material and method : The establishment of blood deficiency model mice by cyclophosphamide state by intraperitoneal injection of 380mg/kg.According to the random parallel control method, 60 C57BL/6 mice were randomly divided into 6 groups according to the random number table method,Normal group, model group, recombinant human erythropoietin group, angelica polysaccharide group, astragalus polysaccharide group,angelica polysaccharide astragalus polysaccharide group, 10 / group.Experiment 1d,mice were weighed, model group, rhEPP group, angelica polysaccharide group, astragalus polysaccharide group, angelica polysaccharide of Astragalus polysaccharide group,intraperitoneal injection of cyclophosphamide 380mg/kg - d -1, continuous 3d, normal group normal saline injection.Made will survive after mould 3 d model mice renumbered grouping,eventually, grouped into 10 normal group, model group, the recombinant human erythropoietin group, angelica polysaccharides, astragalus polysaccharides group, angelica sinensis polysaccharide plus astragalus polysaccharides group ,8/group.The 4d of experiment 4 d (Id after building) , the mice were killed by cervical dislocation, femur and tibia were taked in sterile,The bone marrow hematopoietic stem cells were extracted into the medium,and the cell concentration was adjusted to 1 * 105 /mL,Secondly, the intervention drugs were added into the medium,The mixture was then added to the 96 hole plate (200uL) / hole and the 24 hole plate (1L/ hole), each set of 3 holes, the surrounding holes were added to the corresponding amount of sterile ultra pure water, the cell plates were placed in 5%CO 2,incubated in incubators of 37 degrees,The experimental section Id and 4d (model 1d and model 4d) blood routine observation of mouse peripheral blood ,The cell proliferation rate was observed by MTT assay (Cell culture 1-3d) in experiment 5-7d,Experiment 11d (cell culture 7d)ELISA method was used to detect cytokine EPO, IL-3, IL-6 and RT-PCR method to detect the expression of intracellular transcription factor JAK2 and STAT5.Results :1.model identificationExperiment 3d(model 3d),Model mice appeared diet decline, slow excretion decreased and the stool soft, listlessness, face, eyes, ears, tail hair pale, Peng vertical and less shiny,weight loss etc.;The peripheral blood of mice, the model mice red blood cells, white blood cells, hemoglobin were lower than before modeling and the normal group,That successful replication deficient model.2.Observation on the growth of bone marrow hematopoietic cells in mice.After cell culture 1d, the number of cells was increased under light microscope;After cell culture 3d, a small number of cell colony formation, composed of 8 or so cells, and the cells are smaller, the same shape, round;Cell culture 5d, the number of cells increased significantly,and a large number of cell colony formation, the number of cells per colony is more, the maximum number of cells can reach about 40. Some of the colony cells were uniform in size,uniform in shape and round in shape, which was a colony of red blood cells (CFU-E);Part of the colony is composed of a colorless cell mass, the cells are scattered, there are dense and evacuation type, which is a single lineage hematopoietic progenitor cell colony(CFU-GM).The cells were cultured in 7d, the cell volume and colony decreased, and the cells were dispersed in the medium.3.the effect of Angelica polysaccharide, astragalus polysaccharide and combination therapy on the proliferation of hematopoietic stem cells3.1 hematopoietic stem cell growth curveIn the model group, the growth curve of hematopoietic stem cells showed an inverse S shape, the cell culture was 24h, and the cells grew slowly and cell death;Cell culture 48h, cell growth faster; cell culture 72h, cell growth was similar to the first 24h.The growth curve of hematopoietic stem cells in other groups was approximately S shape, cell culture 24h, rapid proliferation, cell culture 48h, cell growth into logarithmic growth phase, and reached the highest peak;Cell culture 72h, cell growth slowed down, into the stationary phase.3.2 effects of Angelica sinensis polysaccharide, astragalus polysaccharide and combination therapy on proliferation of hematopoietic stem cellsCell culture 24h, 48h and 72h ,MTT method was used to observe the proliferation of cells. The results showed that the hematopoietic stem cells proliferated in different culture systems.Compared with the model group,Normal group, recombinant human erythropoietin group, angelica polysaccharide group, astragalus polysaccharide group, angelica polysaccharide astragalus polysaccharide group increased (P < 0.01);Compared with the normal group,Cell culture after 24h,The cell proliferation of recombinant human erythropoietin (EPO) group, Angelica sinensis polysaccharide group, astragalus polysaccharide group and angelica polysaccharide astragalus polysaccharide group (P < 0.05,P < 0.01), there was no significant difference between the groups (P > 0.05).4.Effects of Angelica sinensis polysaccharide, astragalus polysaccharide and combination therapy on the expression of cytokines EPO, IL-3 and IL-6Cell culture 7d,The expression of cytokines EPO, IL-3 and IL-6 were detected by ELISA method,Results show,The cytokines EPO, IL-3 and IL-6 were expressed in each culture system.Compared with the model group, the expression of cytokines IL-3, EPO and IL-6 in the other groups were significantly higher (P<0.01);Compared with the normal group,the expression of cytokine IL-3 and EPO in the drug intervention group was significantly higher (P<0.01), and the expression of cytokine IL-6 was not significantly different (P >0.05);Compared with the recombinant human erythropoietin group, the expression of IL-3 and EPO in the Radix Angelicae Sinensis and Radix Astragali group was significantly lower(P<0.01), and the expression of cytokine IL-6 was not significantly different (P > 0.05);The expression of cytokines IL-3, EPO and IL-6 were significantly increased in the astragalus polysaccharide group (P<0.01);Compared with Angelica group, 1L-3 group of cytokines astragalus polysaccharide, EPO activity was significantly decreased (P<0.01), cytokine IL-6 expression had no significant difference (P > 0.05); Angelica Polysaccharide of Astragalus polysaccharide group of cytokines IL-3, EPO and IL-6 expression was significantly increased(P<0.01); compared with astragalus polysaccharide group, angelica polysaccharide of astragalus polysaccharide group of cytokines IL-3, EPO, IL-6 expression was significantly increased (P<0.01).5.Effects of Angelica sinensis polysaccharide, astragalus polysaccharide and combination therapy on the expression of JAK2 and STAT5 in the cellsCell culture 7d,RT-PCR method was used to detect the expression of STAT5 and JAK2mRNA in monocytes,The results showed that the transcription factors STAT5 and JAK2 were expressed in the culture system.Compared with the model group, the other 5 groups in cell STAT5, JAK2 expression increased (P<0.05); Compared with the normal group,The drug intervention group The, the expression of cell STAT5 and JAK2 were increased in eath drug intervention group(P<0.01 or P<0.05); Compared with recombinant human erythropoietin,There was no significant difference in the expression of STAT5 and JAK2 in the cell of Angelica sinensis group (P<0.05);The expression of STAT5 and JAK2 in Astragalus group were significantly lower (P<0.01);In angelica polysaccharide astragalus polysaccharide group,the expression of STAT5 was significantly increased (P<0.01), and there was no significant difference in JAK2 expression(P > 0.05);Compared with Angelica polysaccharide group, the expression of STAT5 in Astragalus polysaccharide group was not significantly different (P>0.05). The expression of JAK2 was significantly lower(P<0.05);There was no significant difference in the expression of JAK2 in Astragalus polysaccharide group (P>0.05);The expression of STAT5 was significantly increased(P<0.01);Compared with astragalus polysaccharide group, the expression of JAK2 and STAT5 in Angelica polysaccharide astragalus polysaccharide group increased significantly(P<0.01).Conclusion :1.the effect of Angelica polysaccharide, astragalus polysaccharide and combination on the proliferation of HSCs in miceAngelica polysaccharide, astragalus polysaccharide and combination can promote the proliferation of HSCs in mice, And angelica polysaccharide astragalus polysaccharide combination effect is obvious.2.Effects of Angelica sinensis polysaccharide, astragalus polysaccharide and combination therapy on cytokine expressionAngelica polysaccharide,astragalus polysaccharide and the combination of drugs on cytokines EPO, IL-3 and IL-6 expression were promoted,The effect of Angelica sinensis polysaccharide combined with astragalus polysaccharide was the best.3.Effects of Angelica sinensis polysaccharide, astragalus polysaccharide and combination therapy on the expression of JAK2 and STAT5 in the cellsAngelica polysaccharide, astragalus polysaccharide and combination therapy can stimulate the expression of JAK2 and STAT5 in the cell,The combination of polysaccharide and astragalus polysaccharide was the best.Description of Angelica polysaccharide, blood mechanism of Astragalus polysaccharides may be through the JAK2/STAT5 signaling pathway regulates hematopoietic progenitor / stem cell proliferation and differentiation of erythroid. |
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