| Background and ObjectiveTransplantation of HSC was widely applied to clinical practice of hematopathy. So far,the transplantation of HSC releases a great quantity of HSCs to blood at first by injecting cytokine,and then HSCs were isolated and obtained to be used to transplant by blood cell detachment machine. Cytokine releases HSCs to peripheral blood,at the same time may lead to a series of side effect ,for example immune response,tumor drug resistance,depression of homing ability of HSCs etc. Meanwhile,Due to high-end price of cytokine,many patients who were waiting for transplantion clinical application have to give up treating. Whether we can find a kind of rough material to substitut for cytokine,or to combine with cytokine to decrease venenosus side effect of cytokine is one of hot spots in the field of hematological systemic disease.Our previous experiment indicated that SVP can strengthen immunity of mice with tumors and promote proliferation of WBC in peripheral blood of postradiotherapy mice.Our study use M-NFS-60 cell line which was used to study hematopoietic system function widely and bone marrow cells as research objects,observe the accelerating-effects of SVP on bone hematopoietic cell proliferation,and study its possible mechanisms and signal pathways.Materials and Methods1 Materials 1.1 SPF BALB/C mice,male or female,5-6 w, (20±2)g weight。1.2 Cell line: r-h-MCSF-depended M-NFS-60(promyelocytic leukemic cell),ordered from ATCC of USA(CRL-1838TM).1.3 SVP:purificated by our research group,including SVPⅣ,SVPA2,SVPA3,SVPA4,SVPA5,SVPⅡ3(referred to asⅣ,A2,A3,A4,A5,Ⅱ3)。2 Methods2.1 Effect of SVPⅣon proliferation, morphology, and receptor expression of mice bone marrow cells .2.1.1 Study of effect of scorpion venom peptideⅣon promoting bone marrow cells proliferation in normal mice. Methylcellulose semisolid colony culture was adopted ,experiment was divided into six groups:blank control group,cytokines(IL-3+M-CSF) group,low dosage SVPⅣgroup,high dosage SVPⅣgroup,low dosage SVPⅣcombination with cytokines group,high dosage SVPⅣcombination with cytokines group . Bone marrow cells were cultured with methylcellulose culture assay,on day 7,11 and 14,the number of CFU was calculated.2.1.2 Observation of morphology of CFU: cells were stained by Wright's-Giemsa's dye to identify cellular type.2.1.3 Fluorescence intensity of IL-3R of CFU on day 14:bone marrow cells were cultured for 14 day,and then immumofluorescence method was used to detect fluorescence intensity of IL-3 receptor.2.2 Study of effect of scorpion venom peptideⅣon CD34+ cell number and expression levels of IL-3 receptor and M-CSF receptor:BALB/C mice were divided into 4 groups randomly: normal sodium control group,cytokine(IL-3+M-CSF) group,SVPⅣgroup(0.4mg/kg,1/10 of LD50),SVPⅣcombination with cytokines (IL-3+M-CSF) group.4 groups mice were intraperitoneal injected normal sodium,SVPⅣand cytokine for 1,3,5 day continuously.2.2.1 Flow cytometry was used to detect CD34+ cell number and effect of SVPⅣon IL-3 receptor,M-CSF receptor 1 day after mice were injected .2.2.2 Flow cytometry was used to detect CD34+ cell number and effect of SVPⅣon IL-3 receptor 3 day after mice were injected .2.2.3 Flow cytometry was used to detect CD34+ cell number and effect of SVPⅣ on IL-3 receptor,M-CSF receptor 5 day after mice were injected . 2.3 Study of effect of scorpion venom peptides on STAT5 phosphorylation of M-NFS-60 cell: M-NFS-60 cells were treated with SVPs,and the expression levels of STAT5 phosphorylation were detected by Western blotting.Results:1. Study of effect of scorpion venom peptideⅣon promoting bone marrow cell proliferation:.on day 7, the number of CFU was higher in low dosage SVPⅣgroup than that in high dosage SVPⅣgroup , on day 11 ,14, the number of CFU was much higher in high dosage SVPⅣgroup than that in cytokines group and low dosage SVPⅣgroup (P<0.05 compared with blank control group).The number of CFU was remarkably increased in combined groups among six groups.On day 7, low dosage SVPⅣcombination with cytokines group achieved the maxlmum ,On day 11,14, high dosage SVPⅣgroup combination with cytokines group achieved the maxlmum (P<0.05 compared with blank control group).2. Observation results of CFU morphology indicated cellular type was granulocyte-monocyte mainly except the normal control group.3. Fluorescence intensity of IL-3R of CFU on day 14:immunofluorescence observation indicated 14 day after cells were cultured, numbers of CFU of cytokines group,low dosage SVPⅣgroup,low dosage SVPⅣcombination with cytokines group were more than that of blank control group,and their fluorescence intensities were strengthened compared with blank control group.4. Effect of scorpion venom peptideⅣon CD34+ cell number:1 day after mice were injected ,the number of CD34+ of SVPⅣcombination with cytokines group was increased to the highest,the next was SVPⅣgroup,cytokines group,normal sodium group in turn; 3 day after mice were injected, the number of CD34+ of SVPⅣcombination with cytokines group was decreased,but also the highest, SVPⅣgroup was equal to that of 1 day after mice were injected, the number of CD34+ of cytokines group was slightly increased; 5 day after mice were injected, the number of CD34+ of SVPⅣcombination with cytokines group was a little decreased compared with that of day 3 after mice were injected,but also the highest, the next was SVPⅣgroup, cytokines group,normal sodium group in turn.5. Effect of scorpion venom peptideⅣon the expression level of IL-3 receptor : 1 day after mice were injected, expression levels of IL-3 receptor of cytokines group and SVPⅣcombination with cytokines group were equal and higher than cytokines group,normal sodium group, 3 day after mice were injected, expression level of IL-3 receptor of SVPⅣgroup was the highest, expression level of IL-3 receptor of cytokines group was equal to that of SVPⅣcombination with cytokines group. 5 day after mice were injected, expression level of IL-3 receptor of SVPⅣgroup continue stepping up and was higher than other groups, SVPⅣcombination with cytokines group,cytokines group,normal sodium group in turn.6. Effect of scorpion venom peptideⅣon the expression level of M-CSF receptor: expression levels of M-CSF receptor of cytokines group was the highest and other groups did not detect difference 1 day after mice were injected, 5 day after mice were injected, expression level of M-CSF receptor of SVPⅣcombination with cytokines group was the highest,other groups were not deteted differences.7. M-NFS-60 cells were treated with SVPs,and the expression level of phosphorylation STAT5 was obviously elevated compared with normal sodium group.Conclusion:1. SVPⅣcan promote haematopoietic CFU in vivo,and has a synergistic effect with cytokine;in vitro SVPⅣcan promote the proliferation of CD34+ cells,and can also promote haematogenesis of granulocyte-monocyte mainly .2. SVPⅣcan elevate expression levels of IL-3R, up-regulation the level of phosphorylation of STAT5,which may be associated wih the activation of IL-3 receptor signal transduction pathway. |
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