Research On Membrane Cytotoxicity Of Artesunate, Morphology And Mechanics Of Lymphocyte And Jurkat Cells

This dissertation includes two parts:(1) Research on the membrane cytotocixity of ART; (2) the morphology and elasticity of Jurkat and healthy human lymphocyte cells detect by AFM and combine LSCM and FCM to discuss the connection between elasticity and cytoskeleton.In the first part, the membrane cytotocixity of ART has not been reported recently and integrity of the cell membrane is a basic requirement for maintaining the biological characteristics of a cell. In this part, cell membrane cytotocixity was investigated. CCK-8 assay suggested that ART could significantly suppress the proliferation of Jurkat cells in a dose-dependent manner. With the concentration of ART increase, the cell number decrease was observed by IFM. Changes in morphology and elasticity of Jurkat cells were detected by AFM. At low concentration, the cell membrane appeared undulating. At high concentration, the cells were out of shape, collapsing. The cell height was decreased and the membrane was wizened. The elasticity of Jurkat cells treated with 50μg/ml ART group was lower than the control group. The membrane integrinβ1 expression decreased not obviously. However, cytoskeleton protein under the membrane, the concentration of F-actin decreased obviously, but the Tubulin-βdecreased not obviously. The activities of plasma membrane Ca2+-Mg2+-ATPase and Na+-K+-ATPase were suppressed following ART exposure, meanwhile membrane potential was depolarized, even hyperpolarized. Western-blot was used to detect intracellular Caspase 3 and Cyclin D1 protein level. The Cyclin D1 protein decreased and Caspase 3 protein was regulated. The downregulation of Cyclin D1 may retard the progress of cell cycle. Hence, cellular membrane represented a plausible target for ART-induced death. This research has great significance for the interaction between drug and cells.In the second part, mechanical properties of individual living cells are known to be closely related to the health and function of the human body. Here, AFM was carried out to characterize the morphology and elasticity of human healthy lymphocyte and cancerous Jurkat cells. The whole cell morphology or ultrastructure was obviously different between these two cells. Jurkat cells were found to have an apparent elasticity significantly ower than that of lymphocyte. LSCM was used to investigate their corresponding sub-membrane cytoskeletal structures. Confocal images showed a significant difference in the organization of their sub-membrane actin structures which directly contribute to their difference in cell elasticity. It appeared that lymphocyte cells possess a more pronounced network of actin filaments than that of Jurkat cells. Meanwhile, the concentration of both F-actin and Tubulin-βof lymphocyte cells were higher than Jurkat cells. After treated with Cyt-B, the morphology and elasticity of two cells were changed. The round shape of lymphocyte was changed into gourd shape, and some nanoparticles assembled. The centre of Jurkat cell depressed, the membrane integrality loss, and some pole appeared on the membrane. After treated with Cyt-B, the elasticity of the two cells decreased. It meant that cell elasticity was close related to cytoskeleton protein F-actin and Tubulin-β. We really believe that this technology will be used for cancer detection and opens a door to study the biophysical properties of signaling domains extending from the cell surface to deeper parts of the cell.