Research On B Lymphocyte Morphology And Biomechanics Based On Atomic Force Microscope

In this work, atomic force microscopy, quantum dot labeling, laser scanning confocal microscopy and flow cytometry were used to investigate the biophysical properties (the parameters including morphology, membrane nanostructures, adhension property, elasticity, distribution of membrane receptors and so on) of the human peripheral blood normal and chronic lymphocytic leukemia (CLL) B lymphocytes. According to the experiments, we obtained results:1) Compared with normal B lymphocytes, the CLL B lymphocytes have bigger and higher morphology, and have rougher surface ultrastructure, and have lesser nonspecific force and elasticity. In addition, through the functionalized tips we measured the special force of CD20 antigen-antibody, and which was four times higher than nonspecific force, and the force map showed that CD20 molecules distributed homogeneously on the normal CD20+B lymphocytes. On the contrary, the CD20 molecules distributed heterogenous on B-CLL CD20+B lymphocytes. These results have shown that AFM was a useful means for clinically diagnosis and screening drugs of CLL.2) As time prolonging, from 12h to 72h, the volume, mid-height and nuclear-cytoplasm ratio of CLL-B cells ascended significantly and the pseudopodia and immunological synapse were observed after being treated 24h. In the activation process, nano-structures of cell surface changed from loose, to aggregated and formed microstructure domain. At the same time variation of the size distribution and roughness were exhibited, and the adhension was increased. The changes of the ultrastructures and biophysics of CLL B cell have contributed to understand deeply CLL B cell activation as well as immune signal transmission. The changes have also played an important role in explaining mechanism of immunologic process.3) Compared with resting B lymphocyte, after being treated with the mitogen, pokeweed mitogen, and staphylococcus aureus cowan strain I for 24h, B lymphocytes surface marker molecular CD69 express was increased, and was in the early stage of activation, and their BCR signaling were strengthen, moreover, cytoskeleton come up rearrangement and the expression of key protein F-actin was increased significantly. Finally, it leads to series changes of biophysical properties, including height, diameter and volume morphological change in different extent, and the ultrastructure of membrane obviously displayed proteins gathering. Meanwhile, the adhesion force of B lymphocytes was increased 2 to 3 folds after being stimulated. Our results could provide new information to further understand the B lymphocyte activation process and their structure-function analyses.These results provide certain methods and data value for B lymphocyte research in biology, and clinical, immune and other area.