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Objective To explore the reparation role of recombinant human bone morphogenetic protein-4 mature peptide (rhBMP-4m) on hematopoietic system injury induced by irradiation in mice.Methods Ninety BALB/c mice were randomly divided into normal control group,model group and rhBMP-4m treatment group,with 30 mice in each group.The mice in model group and rhBMP-4m treatment group were irradiated by 60Co γ-ray(7.5 Gy) for 200 s;while the mice in normal control group did not receive irradiation.After irradiation,the mice in model group were given physiological saline 1.0 mL by peritoneal injection per day for 6 days;the mice in rhBMP-4m treatment group were given rhBMP-4m 0.5 mg by peritoneal injection per day for 6 days.The peripheral white blood cell count and marrow mononuclear cells,the percent of CD34 + cells in marrow mononuclear cells of mice were detected on the first,third,fifth,seventh,ninth day after irradiation;the number of spleen nodus and the pleen weight/body weight ratio were detected at the ninth day after irradiation.Results There was no statistic difference in the number of peripheral white blood cells of mice among the three groups on the first day after irradiation(P ＞ 0.05).The peripheral white blood cell count of mice in model group was significantly lower than that in the normal control group on the third,fifth,seventh,ninth day after irradiation (P ＜ 0.01).There was no statistic difference in the number of peripheral white blood cells of mice between rhBMP-4m treatment group and model group on the third,fifth day after irradiation (P ＞ 0.05);the peripheral white blood cell count of mice in rhBMP-4m treatment group was significantly higher than that in model group on the seventh,ninth day after irradiation (P ＜ 0.05).The number of marrow mononuclear cells of mice in model group was significantly lower than that in the normal control group at each time piont after irradiation(P ＜ 0.05).There was no statistic difference in the number of marrow mononuclear cells of mice between the rhBMP-4m treatment group and model group on the first,third day after irradiation (P ＞ 0.05);the number of marrow mononuclear cells of mice in rhBMP-4m treatment group was significantly higher than that in the model group on the fifth,seventh,ninth day after irradiation(P ＜ 0.05).The proportion of CD34 + cells in mononuclear cells of mice in model group was significantly lower than that in the normal control group at each time piont after irradiation(P ＜0.01).There was no statistic difference in the proportion of CD34 + cells in mononuclear cells of mice between the rhBMP-4m treatment group and model group on the first,third day after irradiation (P ＞ 0.05);the proportion of CD34 + cells in mononuclear cells of mice in rhBMP-4m treatment group was significantly higher than that in the model group on the fifth,seventh,ninth day after irradiation(P ＜ 0.05).Compared with the normal control group,the number of spleen nodus was increased and the spleen weight/body weight ratio of mice was decreased in model group on the ninth day after irradiation(P ＜ 0.05);the number of spleen nodus and the spleen weight/body weight ratio of mice in rhBMP-4m treatment group were significantly higher than those in the model group on the ninth day after irradiation (P ＜ 0.01).Conclusion rhBMP-4m can accelerate the reconstruction of bone marrow hematopoietic system of mice with hematopoietic system injury induced by irradiation.
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Objective To study the effects of eukaryotic expressive mature peptide LL-37 of human cationic antimicrobial peptide(hCAP-18) on the expressions of membrane molecules on dendritic cells(DCs).Methods By gene cloning,the eukaryotic expressive plasmid pcDNA4/Myc-His-LL-37 for the mature peptide LL-37 of hCAP-18 was constructed.Then they were transfected into HEK293 cell lines.After the cell lines were cultivated for 48 h,the supernatant was collected.Then the DCs from peripheral blood mononuclear cells(PBMCs) induced by rh-GM-CSF and rh-IL-4 were cultivated with the supernatant for 48h.The expressions of membrane molecules CD40 and HLA-DR on DCs were detected by flow cytometry(FCM).Results The eukaryotic expressive plasmid pcDNA4/Myc-His-LL-37 was constructed successfully and it expressed in eukaryotic cells HEK293.FCM results indicated that the expressions of CD40 and HLA-DR on the membrane of DCs which were stimulated by the supernatant produced by pcDNA4/Myc-His-LL-37 were higher than those in control group(P
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Objective To express the hNGF? gene segment encoding mature peptide (hNGF? ) in E.coli and determine its bioactivity. Methods The resulting gene of hNGF? was subclonedinto the hNGF? site of the expression vector plasmid pBV220. The ligation products were used to transform the competent E.coli DH 5?. The proteins of hNGF? were expressed by temperature induction. The expression products were dealed with solubilizing inclusion bodies and refolding protein. It was introduced into the expressed hNGF? tests of neurite growth of dorsal root knot of chicken embryo and tests of Brdu incorporation into PC12 cells was a biologically active protein. Results The recombinant plasmid pBV220/NGF? was successfully constructed. The NGF? was inserted to pBV220 plasma, a prokaryotic expression vector. Expression of NGF? in E.coli was induced by raising temperature to 42℃. SDS-PAGE electrophoresis showed that NGF? protein existed in inclusion. The solubility protein of NGF? was obtained through purification of inclusion by centrifugation and technique of protein repatriation. Recombinant NGF? protein was purified by affinity chromatography of heparin SepharoseCL-6B. The purity of NGF? was higher than 90% and yield of NGF? was 1.8～2.0mg/L expressing bacteria. The bioactivity of NGF? expressing prokaryotic cell was 1?10 5BU/g according to rule concerning examination of biological products in China. Conclusion The hNGF?gene with bioactivity can be expressed in E.coli.