Visualization Of Mesenchymal Stem Cells Into Osteoblasts And Apoptosis Of Nasopharyngeal Carcinoma Cells Induced By Taxol

This paper includes two parts:(1) The mechanism of hUCMSCs osteogenic differentiation induced by icariin (ICA) was studied by AFM. (2) AFM was used to study the apoptosis of nasopharyngeal carcinoma (CNE-2) cells induced by taxol.In the first part, We verified the hUCMSCs using three methods, such as the morphologic characteristic, surface antigens and the capacity of differentiation into osteoblasts and adipocytes. In order to detect if ICA could induce hUCMSCs to differentiate into osteoblasts, we used the improvemental method of Gomori's calcium-cobalt staining. The most effective concentration was decided by ALP-positive cell counts, then we used the same concentration of ICA to induce the differentiation of hUCMSCs. The result demonstrated that:(1) When the cells were cultured for 7 days, the morphology of cells looked like typical fibroblast by optical microscopy. After cultured for 10 days, the form of cells is polarity arrangement and swirl. (2) The results of FACS indicated that CD29,CD44,CD 105 could express on hUCMSCs which were derived from human unbilical cord, but there were not expression of CD34,CD45 and HLA-DR. (3) ICA could significantly increase the ALP activity of hUCMSCs and promote the mineralization nodules formed (P<0.05).1×10-6mol/L ICA was proved to be the most effective concentration to inducing hUCMSCs into osteoplast. (4) The properties of morphology of ICA and cells were detected by AFM. Images were analyzed using AFM software. Icariin particles were dispersedly distributed on the coverslip and the diameter of particles were around 70±25nm。After cells cultured for 5 days, ICA particles accumulated to micro-domain on cell surface and the diameter of particles increased to 96±21nm.200-300nm micro-pores appeared on the cell membrane when cells were induced by ICA for 10 days. The depth of micro-pore's depth decreased on cell membrane when cells were cultured for 15 days and the cell membrane began to heal. The phenomenon of cellular endocytosis appeared. The undifferentiated hUCMSCs were displayed long spindle shape, while the morphology of differentiatial hUCMSCs changed into square shape. Roughness of the cell membrane was more bigger than that of control cells. Calcium nodules were formed because of osteogenic differentiation, so the surface of cells had obvious small synapses. Atomic force microscope can provide a new tool to study osteogenic differentiation of human umbilical cord mesenchymal stem cells, which is an intuitive approach to study the differentiation of mesenchymal stem cells. In the second part, visualization of nasopharyngeal carcinoma (CNE-2) cell apoptosis was studied by AFM and MTT assay. The main results were the following:(1)Taxol has significant killing effects on the CNE-2 cells, and the MTT assay results indicated that there is a dose-dependent response on CNE-2 cells induced by taxol. The cell activity was inhibited by taxol in a dose-dependent effect, and the cell death can be induced by taxol. (2)Besides, the cytoskeleton was prepared by using TritonX-100. Comparing to normal cells, the volume of apoptosis cell shrinks. (3)As the culture time with taxol increased, the amounts of microvillus decreased significantly, and the average roughness of cell surface increased a lot after taxol treatment. (4)AFM imaging analysis showed that the CNE-2 cell cytoskeleton was damaged by taxol. All these data indicated that atomic force microscope is a visual tool to study the cytoskeleton.