The Study On Mechanical Characteristics Of The Bone Marrow Stem Cells In Mice

Bone marrow stem cells are pluripotent cells derived from bone marrow that can be amplified under appropriate conditions, and holding the potential to organize a series of lineages. For example adipocytes, osteoblasts, chondrocytes, hepatocytes, etc. Both the structure and function of cells will change in the process of induced differentiation. The morphology of the cells was spindle or oval after induced differentiation, and the differentiation of the cells can be tested by using the staining agent and antibody. Due to this results, scientists are started to focus on the research value of bone marrow stem cells. During differentiation, morphology and mechanical properties of stem cells along with the role of regulatory mechanisms and signal transduction also changes. These changes reflected in the cell volume, skeletal structure, morphology and microfilament traction size and distribution. However, the research on bone marrow stem cells mechanical change during differentiation was very limited. We tried to observe the changes of the traction force during the differentiation of bone marrow stem cells, therefore the mechanism of the change of the mechanical properties of stem cells in the process of differentiation was explored.Cell traction forces(CTFs) is one of the most important mechanical parameter of bone marrow stem cells. CTFs are generated by actomyosin interactions and actin polymerization. Once transmitted to the extracellular matrix(ECM) through stress fibers via focal adhesions, which are assemblies of ECM proteins. Previous studies showed that CTFs presents decreased trend both in carcinogenesis and knockout Pinch-3 protein. Moreover, CTFs controlled by intracellular regulatory factors such as alpha-smooth muscle actin ??-SMA). Therefore, select bone marrow stem cells as experiment material and CTFM as experiment method, analysis CTFs, studied and analysis protein and actin cytoskeleton before and after adipogenic and osteogenic differentiation. It aims to explore the mechanical properties evolution and mechanisms of stem cells in the process of induced differentiation.Object: To compare the mechanical index changes of mice bone marrow stem cells by observation and analysis the change of CTFs, ?-SMA protein, and cytoskeleton before and after adipogenic and osteogenic differentiation. To explore the mechanics characteristics of cells in the process of adipogenic and osteogenic differentiation. Methods and conclusions:1.Culture and proliferation the cells. Culture femur and tibia bone marrow of mice, obtained cells by differential adhesion combined with 24-hour first exchange method. Culture cells in CO2 incubator with constant temperature and constant CO2, and subculture. Culture to appropriate concentration,until most cells have good state.2.Measure the traction force distribution, the maximum displacement, the maximum displacement, the maximum traction force and the root mean square traction force before and after adipogenic and osteogenic differentiation by cell traction forces microscopy(CTFM). The results showed that: CTFs increased obviously after adipogenic differentiation, and increased by about 32%. CTFs increased more obviously after osteogenic differentiation, and increased by about 48%. Description Stem cell differentiation is closely related to cell traction.3.Testing of the ?-SMA protein in single cells before and after differentiation by fluorescent antibody staining. The results showed that: the brightness of adipogenic and osteogenic differentiation cells are higher than before, which illustrate that the level of ?-SMA protein were increased after adipogenic and osteogenic differentiation, and the level of ?-SMA protein after osteogenic differentiation is much higher than adipogenic differentiation.4.Testing of the changes of cells cytoskeleton before and after adipogenic and osteogenic differentiation by phalloidin staining. The results showed that: the brightness of adipogenic and osteogenic differentiation cells are decreased, which illustrate that microfilaments became thin, dyeing became light,sparse and irregular arrangement. And the change level of adipogenic differentiation is lower than osteogenic differentiation.5.Testing of the content of total ?-SMA protein before and after adipogenic and osteogenic differentiation by Western blotting. The results showed that: electrophoretic bands widen after adipogenic and osteogenic differentiation, which illustrate that the content of total ?-SMA protein increased after adipogenic and osteogenic differentiation, and the change level of adipogenic differentiation is lower than osteogenic differentiation.