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
AIM： To investigate the association of cyclooxygenase-2 （COX-2） expression with angiogenesis and the number and type of inflammatory cells （macrophages/Kupffer cells; mast cells） within primary hepatocellular car...AIM： To investigate the association of cyclooxygenase-2 （COX-2） expression with angiogenesis and the number and type of inflammatory cells （macrophages/Kupffer cells; mast cells） within primary hepatocellular carcinoma （HCC） tissues and adjacent non-tumorous （NT） tissues. METHODS： Immunohistochemistry for COX-2, CD34, CD68 and mast cell tryptase （MCT） was performed on 14 well-characterized series of liver-cirrhosis-associated HCC patients. COX-2 expression and the number of inflammatory cells in tumor lesions and surrounding liver tissues of each specimen were compared. Moreover, COX-2, CD34 staining and the number of inflammatory cells in areas with different histological degrees within each tumor sample were comparatively analyzed. RESULTS： The percentage of COX-2 positive cells was significantly higher in NT tissues than in tumors. COX-2 expression was higher in well-differentiated HCC than in poorly-differentiated tissues. Few mast cells were observed within the tumor mass, whereas a higher number was observed in the surrounding tissue, especially in peri-portal spaces of NT tissues. Abundant macrophages/ Kupffer cells were observed in NT tissues, whereas the number of cells was significantly lower in the tumor mass. However, a higher cell number was observed in the welldifferentiated tumor and progressively decreased in relation to the differentiation grade. Within the tumor, a positive correlation was found between COX-2 expression and the number of macrophages/Kupffer cells and mastcells. Moreover, there was a positive correlation between CD34 and COX-2 expression in tumor tissues. Comparison between well- and poorly-differentiated HCC showed that the number of CD34-positive cells decreased with dedifferentiation. However, COX-2 was the only independent variable showing a positive correlation with CD34 in a multivariate analysis. CONCLUSION： The presence of inflammatory cells and COX-2 expression in liver tumor suggests a possible relationship with tumor angiogenesis. COX-2 expressing cells and the number of macrophages/Kupffer cells and mast cells decrease with progression of the disease.展开更多
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。
基金Supported by the MIUR and Progetto Strategico Oncologia "Terapia Preclinica Moleculare Oncologia" MIUR-CNR
文摘AIM： To investigate the association of cyclooxygenase-2 （COX-2） expression with angiogenesis and the number and type of inflammatory cells （macrophages/Kupffer cells; mast cells） within primary hepatocellular carcinoma （HCC） tissues and adjacent non-tumorous （NT） tissues. METHODS： Immunohistochemistry for COX-2, CD34, CD68 and mast cell tryptase （MCT） was performed on 14 well-characterized series of liver-cirrhosis-associated HCC patients. COX-2 expression and the number of inflammatory cells in tumor lesions and surrounding liver tissues of each specimen were compared. Moreover, COX-2, CD34 staining and the number of inflammatory cells in areas with different histological degrees within each tumor sample were comparatively analyzed. RESULTS： The percentage of COX-2 positive cells was significantly higher in NT tissues than in tumors. COX-2 expression was higher in well-differentiated HCC than in poorly-differentiated tissues. Few mast cells were observed within the tumor mass, whereas a higher number was observed in the surrounding tissue, especially in peri-portal spaces of NT tissues. Abundant macrophages/ Kupffer cells were observed in NT tissues, whereas the number of cells was significantly lower in the tumor mass. However, a higher cell number was observed in the welldifferentiated tumor and progressively decreased in relation to the differentiation grade. Within the tumor, a positive correlation was found between COX-2 expression and the number of macrophages/Kupffer cells and mastcells. Moreover, there was a positive correlation between CD34 and COX-2 expression in tumor tissues. Comparison between well- and poorly-differentiated HCC showed that the number of CD34-positive cells decreased with dedifferentiation. However, COX-2 was the only independent variable showing a positive correlation with CD34 in a multivariate analysis. CONCLUSION： The presence of inflammatory cells and COX-2 expression in liver tumor suggests a possible relationship with tumor angiogenesis. COX-2 expressing cells and the number of macrophages/Kupffer cells and mast cells decrease with progression of the disease.
