PDF(Pubmed)
Abstract：
UNASSIGNED: Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells.
UNASSIGNED: Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method.
UNASSIGNED: IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells.
UNASSIGNED: For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.
UNASSIGNED: Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method.
UNASSIGNED: IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells.
UNASSIGNED: For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.
摘要：
■肥大细胞与IgE介导的疾病密切相关，例如，过敏和哮喘。人类肥大细胞是异质的，来自不同解剖部位的肥大细胞对某些刺激和药物的反应不同。因此，在建立模型系统时，肥大细胞的起源很重要，和人肺肥大细胞是哮喘研究中高度相关的细胞。因此,我们着手优化IgE介导的人肺肥大细胞活化的方案。
■通过酶消化和机械破坏从接受肺切除术的患者的肺组织中提取人肺肥大细胞，然后进行CD117磁激活细胞分选（MACS）富集。测试了用于IgE介导的脱颗粒的不同培养基和条件以获得优化的方法。
■与用10％血清培养的细胞相比，在无血清培养基中培养的人肺肥大细胞的IgE交联产生了更强的反应。干细胞因子(SCF)的添加不增强脱粒。然而,在加入抗IgE抗体前30分钟将细胞置于新鲜的无血清培养基中，细胞反应更有力。在加入抗IgE后10分钟达到最大脱粒。CD63和CD164都被鉴定为随时间检测脱颗粒肥大细胞的稳定标志物。而抗CD107a和抗生物素蛋白的染色在活化后10分钟开始下降。活化细胞中CD203c和CD13的水平没有变化，因此不能用作人肺肥大细胞的脱颗粒标志物。
■对于最佳脱粒反应，人肺肥大细胞应在无血清培养基中培养和活化。使用这种方法，获得了具有低供体间差异的非常强且一致的脱颗粒反应。因此,该模型可用于进一步研究IgE介导的肥大细胞活化和探索靶向人肺肥大细胞的药物，例如,在哮喘的背景下。
■通过酶消化和机械破坏从接受肺切除术的患者的肺组织中提取人肺肥大细胞，然后进行CD117磁激活细胞分选（MACS）富集。测试了用于IgE介导的脱颗粒的不同培养基和条件以获得优化的方法。
■与用10％血清培养的细胞相比，在无血清培养基中培养的人肺肥大细胞的IgE交联产生了更强的反应。干细胞因子(SCF)的添加不增强脱粒。然而,在加入抗IgE抗体前30分钟将细胞置于新鲜的无血清培养基中，细胞反应更有力。在加入抗IgE后10分钟达到最大脱粒。CD63和CD164都被鉴定为随时间检测脱颗粒肥大细胞的稳定标志物。而抗CD107a和抗生物素蛋白的染色在活化后10分钟开始下降。活化细胞中CD203c和CD13的水平没有变化，因此不能用作人肺肥大细胞的脱颗粒标志物。
■对于最佳脱粒反应，人肺肥大细胞应在无血清培养基中培养和活化。使用这种方法，获得了具有低供体间差异的非常强且一致的脱颗粒反应。因此,该模型可用于进一步研究IgE介导的肥大细胞活化和探索靶向人肺肥大细胞的药物，例如,在哮喘的背景下。
