Hu Jianjiang, Hou Yanming, Zhang Qian, Lei Hongtao, Wang Yi, Wang Danqiao
Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2011 Jul;36(14):1860-4.
To establish a new, real time, dynamic and direct optical detection method for mast cell degranulation caused by anaphylactoid reaction.
A CD63-GFP plasmid was constructed and introduced steadily into rat basophilic leukemia (RBL-2H3) cells. The movements of CD63-GFP, which was located on both the granule membranes and the plasma membranes of RBL cells stimulated by Compound 48/80, were studied by confocal laser scanning microscope (CLSM) and total internal reflection fluorescence microscope (TIRFM) both inside and on the surface of living RBL-2H3 cells.
Before antigen stimulation, most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation, the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.05 micron x s(-1).
Analysis of the movement of each granule provided a new insight into the elementary process of degranulation. The method is rapid, sensitive and reliable, which could be used as a new detection method for anaphylactoid reaction in vitro.
建立一种针对类过敏反应引起的肥大细胞脱颗粒的新型、实时、动态且直接的光学检测方法。
构建CD63 - GFP质粒并稳定导入大鼠嗜碱性白血病（RBL - 2H3）细胞。利用共聚焦激光扫描显微镜（CLSM）和全内反射荧光显微镜（TIRFM），在活的RBL - 2H3细胞内部及表面研究经化合物48/80刺激后位于RBL细胞颗粒膜和质膜上的CD63 - GFP的运动情况。
在抗原刺激前，RBL细胞中大多数带有CD63 - GFP的颗粒几乎不移动。然而，抗原刺激后，颗粒剧烈移动。它们在几分钟内到达质膜并瞬间与之融合。经计算，抗原刺激时颗粒向质膜移动的速度为0.05微米×秒⁻¹。
对每个颗粒运动的分析为脱颗粒的基本过程提供了新的见解。该方法快速、灵敏且可靠，可作为体外类过敏反应的一种新型检测方法。
