We tried to clarify the role of oxygen radicals released from granulocytes stimulated byphorbol myristate acetate(PMA) in rat acute lung injury. It was found that DNA strand-breakdamage(DSBD) in peripheral white blood...We tried to clarify the role of oxygen radicals released from granulocytes stimulated byphorbol myristate acetate(PMA) in rat acute lung injury. It was found that DNA strand-breakdamage(DSBD) in peripheral white blood cells (WBC) was significantly increased 40 min after injec-tion of PMA. DSBD in lung tissue of rats treated with PMA was also markedly increased comparedwith the controls. The PMA-treated rats showed significantly higher lipid-peroxide (LPO) level inplasma and lung tissue hemogenate than the controls did. These results suggest that determination ofDSBD, a simple and sensitive indicator for oxygen radical damaging, might be useful in thediagnosis of adult respiratory distress syndrome (ARDS), when it is used together with themeasurement of plasma LPO.展开更多
Objective:To compare the protein profile of culture supernatants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status.Methods:Dermal...Objective:To compare the protein profile of culture supernatants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status.Methods:Dermal fibroblasts were stimulated with phorbol 12-myristate 13-acetate(PMA)/ionomycine for 72 h.MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern.Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8%–16%.Results:The supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and78.8 k Da.These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 k Da band was appeared in unstimulated and 72 h PMA stimulated cells.Moreover,we found another seven small size(10–19.5 k Da) proteins in supernatants of48 h and 72 h unstimulated but not in PMA or PMA/Ionomycine stimulated fibroblasts.Most of these proteins expression were down regulated following fibroblast activation.This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death.Conclusions:Human fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation.Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay.展开更多
目的了解佛波酯-12-肉豆蔻酸酯-13-乙酸酯（PMA）分化的THP-1（人单核细胞白血病肿瘤细胞）上巨噬细胞标记特征,评估其作为人源巨噬细胞的替代品的可行性。方法用PMA诱导分化THP-1细胞72 h（活化组）,并同步分离随机健康O型献血者的新...目的了解佛波酯-12-肉豆蔻酸酯-13-乙酸酯（PMA）分化的THP-1（人单核细胞白血病肿瘤细胞）上巨噬细胞标记特征,评估其作为人源巨噬细胞的替代品的可行性。方法用PMA诱导分化THP-1细胞72 h（活化组）,并同步分离随机健康O型献血者的新鲜外周血单个核细胞（PBMC）,其中一部分PBMC用人粒细胞-巨噬细胞集落刺激因子（GM-CSF）培养7 d（7 d PBMC）,而未处理的THP-1作为未活化组,分别用流式细胞仪分析这4组细胞的CD14、CD16、HLA-Ⅰ、HLA-DR等细胞表型,并分析Fc受体CD32、CD32b的组成;并用已知含人源HLA-Ⅰ、Ⅱ类抗体、抗-E、健康献血者血浆各1份以及6种未确定抗体特异性但免疫血液学试验检出疑有不规则抗体的血浆与活化THP-1和新鲜PBMC细胞共培养,观察CD14、CD16、HLA-I、HLA-DR等变化;制备O型IgG致敏红细胞和非致敏红细胞,分别用活化THP-1和PBMC细胞与致敏、非致敏红细胞共培养,进行单核细胞单层试验（MMA）。结果观察到THP-1细胞未活化组为CD14~＋CD16^-,活化组为CD14~＋CD16^（＋d）,缺乏PBMC中CD14^-CD16~＋以及非经典CD14^（＋h） CD16~＋,后者在7 d PBMC中明显增加;未活化THP-1表达HLA-Ⅰ类、CD32分子,但HLA-Ⅱ类和CD32b较弱,在PMA刺激后这些抗原或标记分子均升高。发现活化THP-1细胞CD14、CD16分子可被人源性血浆吸附而致下降,而6例未确定抗体特异性的血浆有4例明显降低HLA-Ⅰ类表达。活化THP-1和PBMC均可成功作为单核细胞单层试验的反应细胞,诱导红细胞调理性吞噬。结论 THP-1可替代人单核巨噬细胞作为人源PBMC来源单核巨噬细胞的替代,细胞群落比较少;但是需要注意其与PBMC来源单核巨噬细胞的不同,并具有HLA抗原特异性,因此其应用范围可能比较局限。展开更多
文摘We tried to clarify the role of oxygen radicals released from granulocytes stimulated byphorbol myristate acetate(PMA) in rat acute lung injury. It was found that DNA strand-breakdamage(DSBD) in peripheral white blood cells (WBC) was significantly increased 40 min after injec-tion of PMA. DSBD in lung tissue of rats treated with PMA was also markedly increased comparedwith the controls. The PMA-treated rats showed significantly higher lipid-peroxide (LPO) level inplasma and lung tissue hemogenate than the controls did. These results suggest that determination ofDSBD, a simple and sensitive indicator for oxygen radical damaging, might be useful in thediagnosis of adult respiratory distress syndrome (ARDS), when it is used together with themeasurement of plasma LPO.
基金Supported by the Research and Technology Council of Babol University of Medical Sciences with Grant No.9031029
文摘Objective:To compare the protein profile of culture supernatants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status.Methods:Dermal fibroblasts were stimulated with phorbol 12-myristate 13-acetate(PMA)/ionomycine for 72 h.MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern.Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8%–16%.Results:The supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and78.8 k Da.These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 k Da band was appeared in unstimulated and 72 h PMA stimulated cells.Moreover,we found another seven small size(10–19.5 k Da) proteins in supernatants of48 h and 72 h unstimulated but not in PMA or PMA/Ionomycine stimulated fibroblasts.Most of these proteins expression were down regulated following fibroblast activation.This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death.Conclusions:Human fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation.Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay.
文摘目的了解佛波酯-12-肉豆蔻酸酯-13-乙酸酯（PMA）分化的THP-1（人单核细胞白血病肿瘤细胞）上巨噬细胞标记特征,评估其作为人源巨噬细胞的替代品的可行性。方法用PMA诱导分化THP-1细胞72 h（活化组）,并同步分离随机健康O型献血者的新鲜外周血单个核细胞（PBMC）,其中一部分PBMC用人粒细胞-巨噬细胞集落刺激因子（GM-CSF）培养7 d（7 d PBMC）,而未处理的THP-1作为未活化组,分别用流式细胞仪分析这4组细胞的CD14、CD16、HLA-Ⅰ、HLA-DR等细胞表型,并分析Fc受体CD32、CD32b的组成;并用已知含人源HLA-Ⅰ、Ⅱ类抗体、抗-E、健康献血者血浆各1份以及6种未确定抗体特异性但免疫血液学试验检出疑有不规则抗体的血浆与活化THP-1和新鲜PBMC细胞共培养,观察CD14、CD16、HLA-I、HLA-DR等变化;制备O型IgG致敏红细胞和非致敏红细胞,分别用活化THP-1和PBMC细胞与致敏、非致敏红细胞共培养,进行单核细胞单层试验（MMA）。结果观察到THP-1细胞未活化组为CD14~＋CD16^-,活化组为CD14~＋CD16^（＋d）,缺乏PBMC中CD14^-CD16~＋以及非经典CD14^（＋h） CD16~＋,后者在7 d PBMC中明显增加;未活化THP-1表达HLA-Ⅰ类、CD32分子,但HLA-Ⅱ类和CD32b较弱,在PMA刺激后这些抗原或标记分子均升高。发现活化THP-1细胞CD14、CD16分子可被人源性血浆吸附而致下降,而6例未确定抗体特异性的血浆有4例明显降低HLA-Ⅰ类表达。活化THP-1和PBMC均可成功作为单核细胞单层试验的反应细胞,诱导红细胞调理性吞噬。结论 THP-1可替代人单核巨噬细胞作为人源PBMC来源单核巨噬细胞的替代,细胞群落比较少;但是需要注意其与PBMC来源单核巨噬细胞的不同,并具有HLA抗原特异性,因此其应用范围可能比较局限。
