Study On The Mechanism Of Astragaloside Ⅳ And Granary Isoflavones In Controlling Bone Marrow Stem Cells Induced By Chemotherapeutic Bone Marrow

Purpose:From the aspect of cell proliferation, hematopoietic factors, JAK2 /STAT5 signal transduction, cell cycle, apoptosis and cell differentiation, explore Astragaloside, Calycosin chemotherapy and bone marrow suppression compatibility protective effect of bone marrow stem cells and mouse Regulatory mechanism.Material and method: 1 C57BL/6 mice 20, male and female, 28~32g, purchased from Beijing Vital River Experimental Animal qualifiers SCXK(Beijing) 2012-0001.l week before the one-time purchase of the experiment, regular feeding one week to adapt to the environment, to give free access to water and food. Referring to the literature, for 3 consecutive days to mice by intraperitoneal injection of cyclophosphamide 380 mg /kg established chemotherapy bone marrow suppression model. After modeling, to the removal of cervical law mice in each group were sacrificed, remove the femur of mice, muscle and connective tissue removed, cut the ends of the femur, with 6 needles with saline repeatedly washed bone marrow cavity, and the red break out of the bone marrow, then filtered needles 4, made in a single cell suspension, 2000 rpm, the supernatant was centrifuged on a centrifuge after 10 minutes, cold 70% ethanol was added 4m L fixed, shaken on a shaker, made bone marrow stem cell sample. Cells were divided into six groups: normal group, model group,“EPO+(G-CSF)”control group, Astragaloside group Calycosin group, two drug compatibility group.Determination and dose regimen:determine Astragaloside administration of 0.2mg/ml through pre-experiment, Calycosin 0.01mg/ml, EPO 50u/ml,(G-CSF)50u/ml, a direct role in the administration bone marrow stem cells in vitro culture system and cultured for 3 days, the indoor 18~20℃,20% relative humidity, 5% CO2,37℃ incubator. 2 MTT assay of bone marrow stem cell growth and proliferation. 3 Using the ELISA assay of bone marrow stem cells, EPO, TGF-β, IL-6 and IL-4protein content of. 4 Western blotting using Western-blot assay and protein expression p JAK2 p STAT5, the use of JAK2, STAT5, m RNA expression was detected by RT-q PCR in bone marrow stem cells of SOCS3. 5 By ELISA assay of bone marrow stem cells CDK4 protein content, using RT-q PCR assay of bone marrow stem cells Foxo, m RNA expression of Cyclin D1. 6 By ELISA assay of bone marrow stem cells caspase3 protein content, using RT-q PCR assay of bone marrow stem cells BCL-xl, m RNA expression of BCL-2 and Bax. 7 Using Western-blot assay protein expression GATA1, using m RNA expression was detected by RT-q PCR in bone marrow stem cells to KLF1, BCL11 A and the NF-E2.Results: 1 two medicine astragalus glycosides,calycosin separately and compatibility of chemotherapy bone marrow suppression in mice bone marrow stem cells proliferation and hematopoietic factor 1.1 astragalus glycosides,calycosin separately two medicine compatibility of chemotherapy and bone marrow inhibition of proliferation of bone marrow stem cells in mice 1.1.1 for 24 h after bone marrow stem cell growth and proliferation Compared with model group, each group had significant difference( P <0.05).Comparison between each treatment group, compared with group calycosin separately,astragalus armour nucleoside group, the two drug compatibility group had significant difference(P<0.05);Compared with the control group, the compatibility of astragalus glycosides group, two groups had no significant difference(P>0.05). 1.1.2 for 48 h after bone marrow stem cell growth and proliferation Compared with model group,each group had significant difference(P<0.05).Comparison between the treatment group,calycosin separately, compatibility of astragalus glycosides group, two groups had no significant difference(P>0.05).Compared with control group, calycosin separately groupthere were significant differences(P<0.05).Compared with the control group,the compatibility of astragalus glycosides group, two groups had no significant difference(P>0.05). 1.1.3 for 72 h after bone marrow stem cell growth and proliferation Compared with model group,each group had significant difference(P<0.05).The comparison between treatment group,compared with group calycosin separately, compatibility of astragalus glycosides group, two groups have significant difference(P<0.05).Compared with control group, the root of remembranous milk vetch a nucleoside group, the compatibility of two medicines group and the control group had no significant difference(P>0.05). 1.2 astragalus glycosides,calycosin separately and the compatibility of chemotherapy the two drugs inhibit mice bone marrow stem cells in bone marrow hematopoietic factor EPO,TGF-β,IL-6 and IL-4 the effect of protein content 1.2.1 effects on EPO protein content Compared with model group,each group had significant difference(P<0.05). Comparison between treatment group,calycosin separately,astragalus glycosides group,between the two drug compatibility groups of three groups of both two more significant differences(P<0.05).Compared with control group,calycosin separately group no significant difference(P>0.05). 1.2.2 affect TGF-β protein content Compared with model group,each group have the significant difference(P<0.05).Comparison between treatment group,calycosin separately, astragalus glycosides group,between the two drug compatibility groups of three groups of both two more significant differences(P<0.05). 1.2.3 effects on IL-6 protein content Compared with model group,each group have the significant difference(P<0.05).Comparison between treatment group,calycosin separately, astragalus glycosides group,two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05). The influence of the pathogen of IL-4 protein contentCompared with model group,each group have the significant difference(P<0.05).Comparison between treatment group, calycosin separately, astragalus glycosides group, between the two drug compatibility groups of three groups of both two more significant differences(P<0.05). Two astragalus glycosides,calycosin separately and drug compatibility sex bone marrow inhibition of mouse bone marrow stem cells to chemotherapy in JAK2/STAT5 signal transduction pathways 2.1 the JAK2 m RNA expression Compared with model group,each group have the significant difference(P<0.05).Comparison between treatment group, calycosin separately, astragalus glycosides group, two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group, the astragalus glycosides group, rui isoflavones,two drug compatibility group had no significant difference(P>0.05). 2.2 the STAT5 m RNA expression Compared with model group, each group have the significant difference(P<0.05).Comparison between treatment group,calycosin separately, astragalus glycosides group,two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group,the astragalus glycosides group, rui isoflavones two drug compatibility group had no significant difference(P>0.05). 2.3 impact on SOCS3 m RNA expression Compared with model group,each group have the significant difference(P<0.05).Comparison between treatment group,calycosin separately, astragalus glycosides group,two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group,the astragalus glycosides group,rui isoflavones,two drug compatibility group had no significant difference(P>0.05). 2.4 impact on p JAK2 protein expression Compared with model group,each group have the significant difference(P<0.05).Comparison between treatment group,calycosin separately, astragalus glycosides group,two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group,calycosin separately,two drug compatibility group had no significant difference(P>0.05). 2.5 impact on p STAT5 protein expression Compared with model group,each group have the significant difference(P<0.05).Comparison between treatment group, calycosin separately, astragalus glycosides group,two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05). Compared with control group,astragalus glycosides group,calycosin separately,two drug compatibility group had no significant difference(P>0.05). 3 astragalus glycosides,calycosin separately and the compatibility of chemotherapy the two drugs inhibit mice bone marrow stem cells in bone marrow cell cycle regulation factor,cell apoptosis factor, differentiation factor influence 3.1 astragalus glycosides,calycosin separately and the compatibility of chemotherapy the two drugs inhibit mice bone marrow stem cells in bone marrow cell cycle factor Foxo,Cyclin D1 and CDK4 influence 3.1.1 The influence of Foxo m RNA expression Compared with model group,each group have the significant difference(P<0.05).Comparison between comparison between treatment group, treatment group,calycosin separately, astragalus glycosides group, two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group, the astragalus glycosides group,rui isoflavones,two drug compatibility group had no significant difference(P>0.05). 3.1.2 The influence of Cyclin D1 m RNA expression Compared with model group,each group have the significant difference(P<0.05).Comparison between treatment group, calycosin separately, astragalusglycosides group,two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group,the astragalus glycosides group,rui isoflavones,two drug compatibility group had no significant difference(P>0.05). 3.1.3 affect CDK4 protein content Compared with model group, each group have the significant difference(P<0.05).Comparison between treatment group,calycosin separately, astragalus glycosides group, two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group,calycosin separately group no significant difference(P>0.05). 3.2 astragalus glycosides,calycosin separately and the compatibility of chemotherapy the two drugs inhibit mice bone marrow stem cells in bone marrow cell apoptosis factors the BCL-2,the influence of the BCL-xl, Bax and caspase3 3.2.1 influence on BCL-2 m RNA expression Compared with model group,each group have the significant difference(P<0.05).Comparison between treatment group, calycosin separately, astragalus glycosides group, two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group,calycosin separately,astragalus glycosides group,two drug compatibility group had no significant difference(P>0.05). 3.2.2 affect the BCL-xl m RNA expression Compared with model group, each group have the significant difference(P<0.05).Comparison between treatment group, calycosin separately, astragalus glycosides group, two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group,calycosin separately,astragalus glycosides group,two drug compatibility group had no significant difference(P>0.05). 3.2.3 The influence of Bax m RNA expression Compared with model group, each group have the significant difference(P<0.05).Comparison between treatment group, calycosin separately, astragalusglycosides group, two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group,calycosin separately,astragalus glycosides group, two drug compatibility group had no significant difference(P>0.05). 3.2.4 effect on the protein content of caspase3 Compared with model group, each group have the significant difference P<0.05).Comparison between treatment group and astragalus armour nucleoside group,two drug compatibility between three groups had significant difference(P<0.05).Compared with control group, calycosin separately, astragalus glycosides group,both drug compatibility groups had significant difference(P>0.05). 3.3 compatibility of astragalus glycosides,calycosin separately and the two medicines on chemotherapy of bone marrow inhibition of mouse bone marrow stem cells differentiated regulation factor GATA1,KLF1,BCL11 A, NF-E2 the effects 3.3.1 affect GATA1 protein expression Compared with model group, each group have the significant difference(P<0.05).Comparison between treatment group, calycosin separately, astragalus glycosides group, two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group, calycosin separately, astragalus glycosides group, two drug compatibility group had no significant difference(P>0.05). 3.3.2 The influence of KLF1 m RNA expression Compared with model group, each group have the significant difference(P<0.05).Comparison between treatment group, calycosin separately, astragalus glycosides group, two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group, calycosin separately, astragalus glycosides group, two drug compatibility group had no significant difference(P>0.05). 3.3.3 The influence of BCL11 A m RNA expression Compared with model group, each group have the significant difference(P<0.05).Comparison between treatment group, calycosin separately, astragalus glycosides group, two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group, calycosin separately, astragalus glycosides group, two drug compatibility group had no significant difference(P>0.05). 3.3.4 The influence of NF-E2 m RNA expressionCompared with model group, each group have the significant difference(P<0.05). Comparison between treatment group, calycosin separately, astragalus glycosides group, two drug compatibility groups of three groups were no significant difference between the two comparison(P>0.05).Compared with control group,calycosin separately, astragalus glycosides group,two drug compatibility group had no significant difference(P>0.05).