B 细胞膜CD20 抗原的分布与单分子力谱探测

Distribution and force spectroscopy of CD20 antigen-antibody binding on the B cell surface

摘要:

CD20 抗原分子在B 细胞上表达下降是慢性B 淋巴细胞白血病 (B-CLL) 的标志性特征。采用激光扫描共聚焦显微镜 (LSCM) 和量子点标记相结合的方法对正常和B-CLL 外周血CD20⁺B 淋巴细胞膜表面CD20 抗原分子的表达及分布进行了荧光成像。同时，采用原子力显微镜 (AFM) 对CD20⁺B 细胞的形貌及超微结构特征进行了表征，并且将AFM 针尖用生物素化的单克隆抗体进行修饰，对CD20⁺B 细胞表面的CD20 抗原-抗体之间的单分子力谱进行了探测。LSCM 荧光图像显示，B-CLL CD20⁺B 淋巴细胞上CD20 分子的表达量比正常CD20⁺B 淋巴细胞显著降低。AFM 结果显示，B-CLL CD20⁺B 淋巴细胞超微结构比正常的粗糙。力谱结果显示，CD20 抗原-抗体的相互作用力大约是非特异性黏附力的5 倍，CD20 分子在正常CD20⁺B 淋巴细胞膜上分布比较均匀，小部分有聚集现象，反之，在B-CLL CD20⁺B淋巴细胞膜表面分布稀疏。利用以上两种方法能进一步观察到B-CLL 外周血B 淋巴细胞的异常，并在一定程度上解释临床上B-CLL 病人对利妥昔的低反应现象，为针对抗原CD20 的治疗用药选择提供参考。

Abstract:

The lower expression of CD20 antigen molecules on the B cell membrane is the primary characteristic of B-chronic lymphocytic leukemia (B-CLL). In this paper, we combined laser scanning confocal microscopy (LSCM) and quantum dots labeling to detect the expression and distribution of CD20 molecules on CD20⁺B lymphocyte surface. Simultaneously, we investigated the morphology and ultrastructure of the B lymphocytes that belonged to the normal persons and B-CLL patients through utilizing the atomic force microscope (AFM). In addition, we measured the force spectroscopy of CD20 antigen-antibody binding using the AFM tips modified with CD20 antibody. The fluorescent images indicated that the density of CD20 of normal CD20⁺B lymphocytes was much higher than that of B-CLL CD20⁺B cells. The AFM data show that ultrastructure of B-CLL CD20⁺B lymphocytes became more complicated. Moreover, the single molecular force spectroscopy data show that the special force of CD20 antigen-antibody was four times bigger than the nonspecific force between the naked AFM tip and cell surface. The force map showed that CD20 molecules distributed homogeneously on the normal CD20⁺B lymphocytes, whereas, the CD20 molecules distributed heterogenous on B-CLL CD20⁺B lymphocytes. Our data provide visualized evidence for the phenomenon of low-response to rituximab therapy on clinical. Meanwhile, AFM is possible to be a powerful tool for development and screening of drugs for pharmacology use.