Research Of Matrine-induced Apoptosis And Preliminary Mechanism In Human Oral Squamous Cacinoma Cell Tca8113

Objective: Studying of Matrine (Mat) on human oral squamous carcinomas Tca8113cellsin vitro inhibition of proliferation and induction of apoptosis,and to detect the effect ofMatrine on tumor apoptosis protein FAS, FASL, Caspase-3, discussing the possiblemechanism of action to providing experimental basis for matrine against oral squamouscell carcinoma.Methods: Taking human oral squamous carcinoma cell Tca8113cell lines as the researchobject, useing the method of in vitro to divided the cells into positive control group(adding3ug/ml paclitaxel), negative control group (configurating culture medium doesnot contain paclitaxel and matrine), experimental group (adding different concentrationsof0.2,0.4,0.8,1.2mg/ml matrine), culturing for24、48、72h respectively.1. Observingeach cell morphological changes by inverted microscope;2. MTT method was used todetect the proliferation of Tca8113cell activity, calculating each group cell proliferationinhibition rate;3. using conventional HE staining to observe the changes of nucleus andmembrane, differentiated cell necrosis and apoptosis;4. useing the method of AO/EBstaining to observing morphological changes of apoptotic cells;5. Observing the apoptoticcells by Hoechst33258;6. taking transmission electron microscope to observe differentconcentrations in the changes of the ultrastructure of cells;7. The distribution of flowcytometry matrine48h cell cycle;8. by using the immune fluorescenceimmunocytochemistry staining to detect the expression of Matrine on apoptosis proteinCaspase-3protein;9. by using immunocytochemical staining (SABC three step method)to detect the expression of Matrine on apoptosis protein FAS, FASL, Caspase-3protein.Results:1. The control group cells grew well in each time period, with fusiform adherent cells full, bright, clear boundary. Function after different time, the cells become buoyancy,cell shrinkage, strong refraction, cavitation sample changes;2. Effects ofMatrine on human oral squamous cell carcinoma Tca8113cells can inhibit the cellproliferation inhibition,the same time period of cell rate increased with increase of theconcentration,the same concentration of matrine-acting on cells,with the timeincreasing,the the inhibition rate is higher and higher,in a dose and time dependencerelationship;3. Matrine effect after24h, HE staining showed with the increase of theconcentration of the matrine cellular adherent rate gradually reduced, cell formed edge setphenomenon, on the edge of the cell membrane formation of crescent shape, evenapoptosis body characteristic of apoptosis;4. Cells after AO/EB staining showed that thenegative control group issued a green fluorescent, good adherent cells, cells with uniformsize, and can see a orange RNA dyeing; In the experimental group with the increase ofconcentration, the cell fluorescence from green to orange yellow and orange of excessivegradually, the nucleus appear nuclear pyknosis, karyorrhexis, and apoptotic bodies;5.Matrine effect after48h, Hoechst33258staining showed that the cells of control grouppresents circular uniform blue fluorescence, boundary clear, uniform color, theexperimental group cells showed nuclear chromatin pyknosis, translucent, uneven color;6.Matrine effects in human oral squamous cell carcinomas Tca8113cells after48h electronmicroscope showed that the control group for typical oral squamous carcinoma cells form,has rich intact organelles, cell surface microvilli, the nuclear shape is irregular, nuclearmembrane integrity, cytoplasm into uniform distribution.the experimental nuclearchromatin mild gathered, microvilli reduced or disappeared, intracytoplasmicmitochondria vacuoles sample early apoptosis changes appeared in cytoplasm, the highconcentration group the cell chromatin pyknosis clusters, gathering in a circular bodysurrounding the nuclear membrane formed the typical characteristic of apopotosis;7.Matrine effects in oral squamous cell carcinoma Tca8113cells after48h, using flowcytometry instrument testing, found that cell proliferation is mainly distributed inthe G0/G1phase, S phase and G2/M phase ratio gradually reduce;8. Immunofluorescence staining showed that, with the increase of matrine concentration,green fluorescent spots appeared in the cells increased gradually, and with the increasingconcentration of fluorescent green spot gradually increased, in a dose dependentrelationship;9. using dyeing by immunocytochemistry SABC three-step method, underthe microscope you can see with the increase of concentration, FAS and Caspase-3protein expression gradually increase,in a dose dependent relationship,however, FASLprotein with matrine dose was increased, the expression has no obvious change.Conclusion:1. Effects of matrine in vitro proliferation of human oral squamous cancercells Tca8113cells have obvious inhibitory effect, in a dose and time dependencerelationship;2. Matrine can induce human oral squamous cell carcinomas Tca8113cellsapoptosis, in a dose and time dependence relationship;3. Matrine can make Tca8113cellswere arrested in G0/G1phase;4. Matrine can raise the expression of Tca8113in tumorcell apoptosis protein FAS and protein Caspase-3, so as to inhibiting the proliferation oftumor cells;5. Matrine may not by lowering the expression of FASL protein, reduce tumorescape and inhibiting tumor cell proliferation;6. Matrine can inhibit human oral squamouscell carcinomas Tca8113cells proliferation activity, induction of apoptosis, increasing theexpression of protein FAS and protein Caspase-3, activating intracellular endonuclease,inducing tumor cell apoptosis.