Research On Stem Cellular Differentiation And Melanoma Cellular Apoptosis Based On Atomic Force Microscopy

The cellular differentiation is an important stage in individual development and growth. The differentiation of endoderm cell leads to tissue and system formation and organogenesis. The abnormal differentiation of cell can cause cancer. Senescence and apoptosis of cell also has a very close relationship with human health. In order to better understand and explore the process and mechanism of normal cell differentiation and tumor cell apoptosis, we use atomic force microscopy (AFM), laser scanning confocal microscopy (LSCM), flow cytometry (FCS), transmission electron microscopy (TEM), CCK-8, western-blot and other methods to study the cells. The surface morphology, internal structure, mechanical properties and the related protein of adipocyte precursor cells, porcine spermatogonial stem cells and mouse melanoma cells were detected during the process of cell differentiation or apoptosis. The following results were obtained:1. The changes of surface structure of adipocyte precursor cells were examined during differentiation. There were some holes around the nucleus. The cellular surface became smooth and the roughness of the membrane and the particle size were all decreased. The adhesion force was reduced from (146.33±11.23) PN to (119.95±8.67) PN. The stiffness and Young's modulus of cellular membrane were also reduced about 20%. The results indicate that mature adipocytes are more fluid and stronger deformation. This is the first time to obtain the changes of cellular ultrastructure and mechanical properties after differentiation of adipocyte precursor cells and the results provide new experimental evidence to understand the energy balance and obesity occurrence and diagnosis.2. The pluripotent of SSCs was firstly studied by AFM. After the SSCs differentiated to adipocytes, the roughness, particle size, adhesion force, stiffness and Young's modulus of the membrane were all increased. The cellular morphology was changed from round spheroid to long spindle. The number of intracellular mitochondria, endoplasmic and vacuoles were also increased to provide energy, which was required with the formation of adipocytes. After the complete differentiation, the mature adipocytes'specific molecule PPARy and C/EBPa appeared. These qualitative and quantitative results can improve understanding of stem cells pluripotent.3. After analysis of temozolomide (TMZ) on the apoptosis of B16 cells, the results demonstrated that TMZ could significantly suppress the proliferation of B16 cells in a dose-dependent manner. The TMZ concentration of 0-50μM induced apoptosis and 100-200μM induced cell death. With the concentration of TMZ increase, changes in morphology and nucleus were irreversibly damaged. The membrane potential, intercellular Ca2+ concentration and caspase-3 protein content were accordingly changed. Our study firstly deduced that the cellular membrane and nucleus toxicity may be the mechanism of B16 cell apoptosis.In summary, we use AFM to detect cell morphology, ultrastructure and mechanical properties. Then combined with biotechnology, we characterized the process of differentiation and apoptosis. These visual data provide basis to in-depth study of life activities.