Study On Apoptosis Of Human Liver Cancer SMMC-7721Cells Induced By Vincristine And The Mitochondrial Pathway

Vincristine is the main constituents of Catharanthus roseus, which has good antitumor activity, this paper primarily through experiment to research on inducement of Vincr-istine on inhibit prolife-ration and apoptosis in SMMC-7721cell and its mechanicsms.The experiments using the MTT and SRB assay inhibition of Vincristine on SMMC-7721cells. In research we use inverted microscope, Hoechst33258staining SMMC-7721for fluorescence microscopy of cell morphology changes, PI staining flow cytometry detection of apoptosis rate. Fluo-3/AM staining CLSM detection the effect of Vincristine on Ca2+concentration in SMMC-7721cells. Rhodaminel23staining CLSM detection the effect of Vincristine on△Ψm in SMMC-7721cells. FCM detection the effect of Vincristine on ROS in SMMC-7721cells.The results show that, Vincristine can inhibit SMMC-7721cells. In the experiment, MTT and SRB all showed phycoerythrin on SMMC-7721cell has certain cytotoxicity, and with the phycoerythrin concentration increases inhibition enhances, the SMMC-7721cell IC500.036μmol/L.Through the inverted microscope observation in direct Vincristine human liver cancer SMMC-7721cells after48h, negative control group cells arranged relatively close together, without cell deformation and fragmentation, with the concentration increased cell growth density gradually thinning, cell shrinkage, high concentration group most of cell disruption, cell morphogenesis is complete, adherent to reduce. Description of Vincristine on SMMC-7721cell is apoptosis, and are dose dependent.Hoechst33258staining, observed under fluorescence microscopy, normal cell nucleus is uniformly blue fluorescence, and apoptotic cell nucleus showed a dense dense or chunky concentrated dye fluorescence. The experimental results show that, Vincristine role for SMMC-7721cells after48h cell shape changes with the increase of dosage, appeared phenomenon of apoptosis, the negative control group the results showed that the SMMC-7721cell chromatin uniform, nuclear morphology rules, in dosage group nuclei fragmentation, high dosage group of nuclei showed a dense dense, this suggests that Vincristine can result in SMMC-7721cell morphological change.The experimental use of propidium iodide (PI) single staining by flow cytometry analysis showed that different concentrations of Vincristine role of SMMC-7721cells after48h, have obvious apoptosis peak, each cell apoptosis rate were (8.53±0.17)%,(10.78±0.06)%,(12.04±0.46)-%, as the drug concentration increased, further description of Vincristine inhibition effect on SMMC-7721cells and inducing tumor cell apoptosis related.Different doses (0.016,0.032,0.064μmol/L) of Vincristine makes Laser Confocal scanning microscope inspection found SMMC-7721elevated intracellular calcium ions, and has a measure of dose-dependent after48h.Study on SMMC-7721cell mitochondrial membrane potential found that Vincristine can reduce cell mitochondrial membrane potential and has a measure of dose-dependent; Study on SMMC-7721cell ROS contention found that Vincristine can induce cell ROS contention.To sum up, Vincristine can inhibit the proliferation of SMMC-7721cells and step-up SMMC-7721levels of intracellular ROS, oxidative damage to mitochondria, which leads to mitochondrial bulge,ROS through oxidation mitochondrial membrane, mitochondrial DNA and some important proteins, resulting function of mitochondrial electron transport chain occurs disabili-ty, resulting in decreased mitochondrial membrane potential, mitochondrial membrane permeability changes, cytochrome C and apoptosis correlation factor release. Calcium ion concentration increased, decreased mitochondrial membrane potential will lead to increased mitochondrial Ca2+uptake, mitochondrial calcium overload leading to mitochondrial damage, cyt-c release, activation of caspases, ultimately leading to apoptosis.