Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA fr...Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct.展开更多
Objective To study the pharmacokinetics of a novel recombinant human granulocyte colonystimulating factor (rhG-CSFa) in rats and to determine the proteolytic rates of rhG-CSFa in the whole blood and serum of rats in v...Objective To study the pharmacokinetics of a novel recombinant human granulocyte colonystimulating factor (rhG-CSFa) in rats and to determine the proteolytic rates of rhG-CSFa in the whole blood and serum of rats in vitro. Methods The pharmacokinetics of rhG-CSFa and conventional (wild type,WT) granulocyte colonystimulating factor (G-CSF) were investigated in Sprague-Dawley rats which received either intravenous or subcutaneous injection of rhG-CSFa or WT G-CSF at three different doses (20,50,or 100 μg/kg). The blood samples of rats were collected at multiple time points (from 0.08 to 12 h) and the concentrations of rhG-CSFa and WT G-CSF in serum were determined with a sandwich enzyme-linked immunosorbent assay (ELISA). For the study of proteolytic rates in vitro,the concentrations of rhG-CSFa or WT G-CSF were determined at 3-minute intervals after addition of the respective drug to rat’s whole blood or serum. Results Pharmacokinetic analysis of serum rhG-CSFa or WT G-CSF levels indicated that,at each dose tested,for either route of drug administration,the area under concentration-time curve values and the maximum serum concentration of rhG-CSFa were higher than those of WT G-CSF,and the serum half life of rhG-CSFa was longer than that of WT G-CSF. Subsequent in vitro whole blood and serum stability study showed that the rates of drug degradation in WT G-CSF were 1.8 folds and 1.5 folds higher than those in rhG-CSFa,respectively. Conclusion rhG-CSFa has better serum and whole blood stability in vitro and higher bioavailability in vivo as compared to WT G-CSF.展开更多
The neuroprotective effects of granulocyte colony-stimulating factor in cerebral ischemia/reperfusion injury are currently contentious. The present study examined the effects of subcutaneous injection of recombinant h...The neuroprotective effects of granulocyte colony-stimulating factor in cerebral ischemia/reperfusion injury are currently contentious. The present study examined the effects of subcutaneous injection of recombinant human granulocyte colony-stimulating factor （50 pg/kg） over 5 days in a model of cerebral ischemia/reperfusion with intraluminal filament occlusion in rats. The results indicated that recombinant human granulocyte colony-stimulating factor reduced brain infarct volume following cerebral ischemia/reperfusion injury in rats, down-regulated the expression of caspase-3 mRNA （a key protease for apoptosis in the cerebral ischemia zone）, lowered the rate of neuronal apoptosis in the cerebral ischemia zone, and notably ameliorated neurological function. These results indicate that recombinant human granulocyte colony-stimulating factor has anti-apoptotic effects on neurons following focal cerebral ischemia/reperfusion injury, and exerts neuroprotective effects.展开更多
The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatogra...The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatography (SAX) in the presence of low concentration- of urea. The effect of urea concentration on this refolding process was investigated. The obtained refolded rhG-CSF has a high specific activity of 2.3×108 U/mg, demonstrating that the proteins were completely refolded during the chromatographic process. With only one step by SAX in 40 min, purity and mass recovery of the refolded and purified rhG-CSF were 97% and 43%, respectively.展开更多
Recombinant human granulocyte colony-stimulating factor （rhG-CSF） in inclusion bodies was solubilized by 8 mol/L urea solution and subsequently precipitated by acetone to improve its purity. After that, the precipit...Recombinant human granulocyte colony-stimulating factor （rhG-CSF） in inclusion bodies was solubilized by 8 mol/L urea solution and subsequently precipitated by acetone to improve its purity. After that, the precipitates were solubilized by sodium hydroxide solution containing 2 mol/L urea. Then the solubilized rhG-CSF was passed through a size exclusion chromatography for refolding and extensive purification, and further purified by a weak anion exchange chromatography. The purity and mass recovery of refolded rhG-CSF were 96.5% and 75.6%, respectively. The bioactivity was 8.4x10^7 IU/mg.展开更多
Alveolar bone regeneration has been strongly linked to macrophage polarization.M1 macrophages aggravate alveolar bone loss,whereas M2 macrophages reverse this process.Berberine(BBR),a natural alkaloid isolated and ref...Alveolar bone regeneration has been strongly linked to macrophage polarization.M1 macrophages aggravate alveolar bone loss,whereas M2 macrophages reverse this process.Berberine(BBR),a natural alkaloid isolated and refined from Chinese medicinal plants,has shown therapeutic effects in treating metabolic disorders.In this study,we first discovered that culture supernatant(CS)collected from BBR-treated human bone marrow mesenchymal stem cells(HBMSCs)ameliorated periodontal alveolar bone loss.CS from the BBR-treated HBMSCs contained bioactive materials that suppressed the M1 polarization and induced the M2 polarization of macrophages in vivo and in vitro.To clarify the underlying mechanism,the bioactive materials were applied to different animal models.We discovered macrophage colony-stimulating factor(M-CSF),which regulates macrophage polarization and promotes bone formation,a key macromolecule in the CS.Injection of pure M-CSF attenuated experimental periodontal alveolar bone loss in rats.Colony-stimulating factor 1 receptor(CSF1R)inhibitor or anti-human M-CSF(M-CSF neutralizing antibody,Nab)abolished the therapeutic effects of the CS of BBR-treated HBMSCs.Moreover,AKT phosphorylation in macrophages was activated by the CS,and the AKT activator reversed the negative effect of the CSF1R inhibitor or Nab.These results suggest that the CS of BBR-treated HBMSCs modulates macrophage polarization via the M-CSF/AKT axis.Further studies also showed that CS of BBR-treated HBMSCs accelerated bone formation and M2 polarization in rat teeth extraction sockets.Overall,our findings established an essential role of BBR-treated HBMSCs CS and this might be the first report to show that the products of BBR-treated HBMSCs have active effects on alveolar bone regeneration.展开更多
Objective:To compare the efficacy and safety of pegylated recombinant human granulocyte colony-stimulating factor(PEG-rhG-CSF)for preventive or delayed treatment in neutropenia,completion rate of concurrent chemoradio...Objective:To compare the efficacy and safety of pegylated recombinant human granulocyte colony-stimulating factor(PEG-rhG-CSF)for preventive or delayed treatment in neutropenia,completion rate of concurrent chemoradiotherapy and hospitalization rate in patients with esophageal squamous carcinoma during definitive concurrent chemoradiotherapy.Methods:A total of 70 patients with esophageal squamous carcinoma in Peking University Cancer Hospital from January 2019 to December 2020,who received PEG-rhG-CSF during concurrent chemoradiotherapy,were enrolled in this retrospective analysis.There were 32 patients in the preventive group,and 38 patients in the delayed group.The incidence of neutropenia,completion rate of concurrent chemoradiotherapy and neutropeniarelated hospitalization rate were compared between PEG-rhG-CSF preventive group and delayed group.Results:The incidence of severe neutropenia(Grades 3–4)in all patients was 31.4%.Comparison between preventive group and delayed group showed that the incidence of severe neutropenia was 6.3%and 39.4%(χ^(2)=10.428,P=0.001),respectively.In preventive group,the incidence of severe neutropenia was 3.7%and 20.0%,respectively,for primary prevention and secondary prevention of PEG-rhG-CSF(χ^(2)=12.321,P=0.001).The completion rate of concurrent chemoradiotherapy was 93.8%in the preventive group and 63.2%in the delayed group(χ^(2)=9.220,P=0.002).The incidence of treatment interruption was 25.7%in the whole group,12.5%in the preventive group and 36.8%in the delayed group(χ^(2)=5.389,P=0.020).Seven patients(7/70,10.0%)were hospitalized and treated with intravenous antibiotics for neutropenia,including 1 in the preventive group and 6 in the delayed group(P=0.078).Conclusions:Prophylactic use of PEG-rhG-CSF during concurrent chemoradiotherapy for patients with esophageal squamous carcinoma can effectively reduce the incidence of neutropenia,ensure the safety of treatment,and improve the completion rate of concurrent chemoradiotherapy.展开更多
目的探究重组人粒细胞集落刺激因子(recombinant human granulocyte-colony stimulating factor,rhG-CSF)在治疗儿童化疗性口腔黏膜炎中的应用效果。方法选取2020年1月至2022年6月于笔者医院肿瘤外科化疗的60例化疗性口腔黏膜炎患儿,按...目的探究重组人粒细胞集落刺激因子(recombinant human granulocyte-colony stimulating factor,rhG-CSF)在治疗儿童化疗性口腔黏膜炎中的应用效果。方法选取2020年1月至2022年6月于笔者医院肿瘤外科化疗的60例化疗性口腔黏膜炎患儿,按随机数字表法分为两组,每组30例。对照组采取生理盐水口腔护理,实验组采取rhG-CSF口腔护理。比较两组患儿干预效果、口腔黏膜炎分度、生存质量[健康状况调查简表(36-item short—form,SF-36)]、患儿家长护理满意度。结果实验组总有效率较对照组高,差异有统计学意义(P<0.05)。干预5d,实验组患儿口腔黏膜炎分度结果优于对照组,差异有统计学意义(P<0.05)。干预5d,两组患儿SF-36评分较干预前高,实验组较对照组高,差异有统计学意义(P<0.05)。实验组患儿家长满意度较对照组高,差异有统计学意义(P<0.05)。结论rhG-CSF口腔护理可有效提高儿童化疗性口腔黏膜炎的干预效果,减轻口腔黏膜炎分度,改善患儿生存质量,提高患儿家长护理满意度。展开更多
Background：Steady-state bone marrow （SS-BM） and granulocyte colony-stimulating growth factor-primed BM/peripheral blood stem-cell （G-BM/G-PBSC） are the main stem-cell sources used in allogeneic hematopoietic stem...Background：Steady-state bone marrow （SS-BM） and granulocyte colony-stimulating growth factor-primed BM/peripheral blood stem-cell （G-BM/G-PBSC） are the main stem-cell sources used in allogeneic hematopoietic stem-cell transplantation.Here,we evaluated the treatment effects of SS-BM and G-BM/G-PBSC in human leucocyte antigen （HLA）-identical sibling transplantation.Methods：A total of 226 patients （acute myelogenous leukemia-complete remission 1,chronic myelogenous leukemia-chronic phase 1） received SS-BM,G-BM,or G-PBSC from an HLA-identical sibling.Clinical outcomes （graft-versus-host disease [GVHD],overall survival,transplant-related mortality [TRM],and leukemia-free survival [LFS]） were analyzed.Results：When compared to SS-BM,G-BM gave faster recovery time to neutrophil or platelet （P 〈 0.05）.Incidence of grade Ⅲ-Ⅳ acute GVHD and extensive chronic GVHD （cGVHD） was lower than seen with SS-BM （P 〈 0.05） and similar to G-PBSC.Although the incidence of cGVHD in the G-BM group was similar to SS-BM,both were lower than G-PBSC （P 〈 0.05）.G-BM and G-PBSC exhibited similar survival,LFS,and TRM,but were significantly different from SS-BM （P 〈 0.05）.There were no significant differences in leukemia relapse rates among the groups （P 〉 0.05）.Conclusions：G-CSF-primed bone marrow shared the advantages of G-PBSC and SS-BM.We conclude that G-BM is an excellent stem-cell source that may be preferable to G-PBSC or SS-BM in patients receiving HLA-identical sibling hematopoietic stem-cell transplantation.展开更多
基金the Natural Science Foundationof Fujian Province, China (No. C97067)
文摘Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct.
基金Supported by State Scientific Key Projects for New Drug Research and Development (2009ZX09102-250)High-tech Research Project for Medicine and Pharmacology of Jiangsu province (BG20070605)
文摘Objective To study the pharmacokinetics of a novel recombinant human granulocyte colonystimulating factor (rhG-CSFa) in rats and to determine the proteolytic rates of rhG-CSFa in the whole blood and serum of rats in vitro. Methods The pharmacokinetics of rhG-CSFa and conventional (wild type,WT) granulocyte colonystimulating factor (G-CSF) were investigated in Sprague-Dawley rats which received either intravenous or subcutaneous injection of rhG-CSFa or WT G-CSF at three different doses (20,50,or 100 μg/kg). The blood samples of rats were collected at multiple time points (from 0.08 to 12 h) and the concentrations of rhG-CSFa and WT G-CSF in serum were determined with a sandwich enzyme-linked immunosorbent assay (ELISA). For the study of proteolytic rates in vitro,the concentrations of rhG-CSFa or WT G-CSF were determined at 3-minute intervals after addition of the respective drug to rat’s whole blood or serum. Results Pharmacokinetic analysis of serum rhG-CSFa or WT G-CSF levels indicated that,at each dose tested,for either route of drug administration,the area under concentration-time curve values and the maximum serum concentration of rhG-CSFa were higher than those of WT G-CSF,and the serum half life of rhG-CSFa was longer than that of WT G-CSF. Subsequent in vitro whole blood and serum stability study showed that the rates of drug degradation in WT G-CSF were 1.8 folds and 1.5 folds higher than those in rhG-CSFa,respectively. Conclusion rhG-CSFa has better serum and whole blood stability in vitro and higher bioavailability in vivo as compared to WT G-CSF.
文摘The neuroprotective effects of granulocyte colony-stimulating factor in cerebral ischemia/reperfusion injury are currently contentious. The present study examined the effects of subcutaneous injection of recombinant human granulocyte colony-stimulating factor （50 pg/kg） over 5 days in a model of cerebral ischemia/reperfusion with intraluminal filament occlusion in rats. The results indicated that recombinant human granulocyte colony-stimulating factor reduced brain infarct volume following cerebral ischemia/reperfusion injury in rats, down-regulated the expression of caspase-3 mRNA （a key protease for apoptosis in the cerebral ischemia zone）, lowered the rate of neuronal apoptosis in the cerebral ischemia zone, and notably ameliorated neurological function. These results indicate that recombinant human granulocyte colony-stimulating factor has anti-apoptotic effects on neurons following focal cerebral ischemia/reperfusion injury, and exerts neuroprotective effects.
基金This work is supported by the National Natural Science Foundation of China(No．20175016)
文摘The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatography (SAX) in the presence of low concentration- of urea. The effect of urea concentration on this refolding process was investigated. The obtained refolded rhG-CSF has a high specific activity of 2.3×108 U/mg, demonstrating that the proteins were completely refolded during the chromatographic process. With only one step by SAX in 40 min, purity and mass recovery of the refolded and purified rhG-CSF were 97% and 43%, respectively.
基金This work is supported by the National Natural Science Foundation of china （No. 20175016 and No. 20475042） the Foundation of Key Laboratory of Modem Separation Science in Shaanxi Province （No. 05JS61）.
文摘Recombinant human granulocyte colony-stimulating factor （rhG-CSF） in inclusion bodies was solubilized by 8 mol/L urea solution and subsequently precipitated by acetone to improve its purity. After that, the precipitates were solubilized by sodium hydroxide solution containing 2 mol/L urea. Then the solubilized rhG-CSF was passed through a size exclusion chromatography for refolding and extensive purification, and further purified by a weak anion exchange chromatography. The purity and mass recovery of refolded rhG-CSF were 96.5% and 75.6%, respectively. The bioactivity was 8.4x10^7 IU/mg.
基金supported by the CAMS Innovation Foundation for Medical Sciences(2016-I2M1-011)A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions(2018-87)+1 种基金Jiangsu Province Capability Improvement Project through Science,Technology and Education-Jiangsu Provincial Research Hospital Cultivation Unit(YJXYYJSDW4)Jiangsu Provincial Medical Innovation Center(CXZX202227)。
文摘Alveolar bone regeneration has been strongly linked to macrophage polarization.M1 macrophages aggravate alveolar bone loss,whereas M2 macrophages reverse this process.Berberine(BBR),a natural alkaloid isolated and refined from Chinese medicinal plants,has shown therapeutic effects in treating metabolic disorders.In this study,we first discovered that culture supernatant(CS)collected from BBR-treated human bone marrow mesenchymal stem cells(HBMSCs)ameliorated periodontal alveolar bone loss.CS from the BBR-treated HBMSCs contained bioactive materials that suppressed the M1 polarization and induced the M2 polarization of macrophages in vivo and in vitro.To clarify the underlying mechanism,the bioactive materials were applied to different animal models.We discovered macrophage colony-stimulating factor(M-CSF),which regulates macrophage polarization and promotes bone formation,a key macromolecule in the CS.Injection of pure M-CSF attenuated experimental periodontal alveolar bone loss in rats.Colony-stimulating factor 1 receptor(CSF1R)inhibitor or anti-human M-CSF(M-CSF neutralizing antibody,Nab)abolished the therapeutic effects of the CS of BBR-treated HBMSCs.Moreover,AKT phosphorylation in macrophages was activated by the CS,and the AKT activator reversed the negative effect of the CSF1R inhibitor or Nab.These results suggest that the CS of BBR-treated HBMSCs modulates macrophage polarization via the M-CSF/AKT axis.Further studies also showed that CS of BBR-treated HBMSCs accelerated bone formation and M2 polarization in rat teeth extraction sockets.Overall,our findings established an essential role of BBR-treated HBMSCs CS and this might be the first report to show that the products of BBR-treated HBMSCs have active effects on alveolar bone regeneration.
文摘Objective:To compare the efficacy and safety of pegylated recombinant human granulocyte colony-stimulating factor(PEG-rhG-CSF)for preventive or delayed treatment in neutropenia,completion rate of concurrent chemoradiotherapy and hospitalization rate in patients with esophageal squamous carcinoma during definitive concurrent chemoradiotherapy.Methods:A total of 70 patients with esophageal squamous carcinoma in Peking University Cancer Hospital from January 2019 to December 2020,who received PEG-rhG-CSF during concurrent chemoradiotherapy,were enrolled in this retrospective analysis.There were 32 patients in the preventive group,and 38 patients in the delayed group.The incidence of neutropenia,completion rate of concurrent chemoradiotherapy and neutropeniarelated hospitalization rate were compared between PEG-rhG-CSF preventive group and delayed group.Results:The incidence of severe neutropenia(Grades 3–4)in all patients was 31.4%.Comparison between preventive group and delayed group showed that the incidence of severe neutropenia was 6.3%and 39.4%(χ^(2)=10.428,P=0.001),respectively.In preventive group,the incidence of severe neutropenia was 3.7%and 20.0%,respectively,for primary prevention and secondary prevention of PEG-rhG-CSF(χ^(2)=12.321,P=0.001).The completion rate of concurrent chemoradiotherapy was 93.8%in the preventive group and 63.2%in the delayed group(χ^(2)=9.220,P=0.002).The incidence of treatment interruption was 25.7%in the whole group,12.5%in the preventive group and 36.8%in the delayed group(χ^(2)=5.389,P=0.020).Seven patients(7/70,10.0%)were hospitalized and treated with intravenous antibiotics for neutropenia,including 1 in the preventive group and 6 in the delayed group(P=0.078).Conclusions:Prophylactic use of PEG-rhG-CSF during concurrent chemoradiotherapy for patients with esophageal squamous carcinoma can effectively reduce the incidence of neutropenia,ensure the safety of treatment,and improve the completion rate of concurrent chemoradiotherapy.
文摘Background：Steady-state bone marrow （SS-BM） and granulocyte colony-stimulating growth factor-primed BM/peripheral blood stem-cell （G-BM/G-PBSC） are the main stem-cell sources used in allogeneic hematopoietic stem-cell transplantation.Here,we evaluated the treatment effects of SS-BM and G-BM/G-PBSC in human leucocyte antigen （HLA）-identical sibling transplantation.Methods：A total of 226 patients （acute myelogenous leukemia-complete remission 1,chronic myelogenous leukemia-chronic phase 1） received SS-BM,G-BM,or G-PBSC from an HLA-identical sibling.Clinical outcomes （graft-versus-host disease [GVHD],overall survival,transplant-related mortality [TRM],and leukemia-free survival [LFS]） were analyzed.Results：When compared to SS-BM,G-BM gave faster recovery time to neutrophil or platelet （P 〈 0.05）.Incidence of grade Ⅲ-Ⅳ acute GVHD and extensive chronic GVHD （cGVHD） was lower than seen with SS-BM （P 〈 0.05） and similar to G-PBSC.Although the incidence of cGVHD in the G-BM group was similar to SS-BM,both were lower than G-PBSC （P 〈 0.05）.G-BM and G-PBSC exhibited similar survival,LFS,and TRM,but were significantly different from SS-BM （P 〈 0.05）.There were no significant differences in leukemia relapse rates among the groups （P 〉 0.05）.Conclusions：G-CSF-primed bone marrow shared the advantages of G-PBSC and SS-BM.We conclude that G-BM is an excellent stem-cell source that may be preferable to G-PBSC or SS-BM in patients receiving HLA-identical sibling hematopoietic stem-cell transplantation.
