The Experimental Study Of Human Tongue Carcinoma Tca8113 Cells Following Photodynamic Therapy Mediated By TMPyP4

Objective: PARTⅠ:To determination the wavelength of Meso-5,10,15,20-Tetrakis-(N-methyl-4-pyridyl) porphine (TMPyP4 ) in different solution. PARTⅡ: To investigate the effect of photodynamic therapy(PDT) mediated by TMPyP4 (TMPyP4-PDT) on Tca8113 cells in vitro, and study the cell death mode.Methods: PARTⅠ:1 TMPyP4 was weighted in the electronic scale under aseptic conditions,and 1mg was removed twice.Then they were diluted in 100ml double distilled water and RPMI-1640 medium which containing 10% fetal calf serum respectively,so that TMPyP4 concentrations in the solutions were the same(about 6.5μM),the electronic absorption wavelenghth and absorbance values of TMPyP4 were determinated at room temperature using UV spectrophotometer UV-265 within the 360nm～700nm.2 Choose the wavelenghth with the largest absorbance value stimulating the 6.5μM TMPyP4 concentration of RPMI-1640 medium which containing 10% fetal calf serum,the excitation wavelength and fluorenscence intensity were tested. PARTⅡ: 1 Tca8113 cells were incubated in RPMI-1640 medium containing 10% fetal calf serum after resuscitation. Cell grew under water-satured sterile atmosphere containing 5% CO2 at 37oC and was assayed for growth curve by means of MTT.2 Tca8113 cells in exponential growth stage were detached by 0.25% pancreatin. Cell suspensions were seeded into 96-well tissue culture plates and allowed to reattach overnight. Then Tca8113 cells were incubated in RPMI-1640 containing serial concentration of TMPyP4 and irradiated with different light energy. Phototoxic effects were analyzed by MTT assay.3 Cells were obvserved in inversed micrcscope after different treatment,then detached by 0.25% pancreatin,the cells were treated according to the instruction.Cell death mode was investigated by flow cytometer using Annexin V/PI doubl-staining method, and others were detected by HE staining method.Result: PARTⅠ:TMPyP4 in double distilled water showed only single-absorption peak,which is at 421.6nm.TMPyP4 in RPMI-1640 medium which containing 10% fetal calf serum showed double-absorption peak,which were at 422.8nm and 557.8nm,and the absorption peak at 557.8nm is significantly higher than that at 422.8nm.For the wavelength of the laser used in the experiment just have 470nm,580nm and 630nm,so the wavelength of 580nm was selected according to the above test.Excited Under the wavelength of 580nm,the maximum absorption peak occurred at the same wavelength. PARTⅡ:1 Simple PDT has no phototoxic effect on Tca8113 cells.2 Different killing efficacy follows different TMPyP4 concentration under different energy of PDT. The anti-cells curve showed"S"shape.Under definite condition,the inhibition rate and induced cell apoptosis rate mediated by TMPyP4-PDT increased with the increase of the dosage and the light enerage.3 The major cell death mode is apoptosis with low concentration of TMPyP4 and mild dosage of PDT and necrosis with high concentration of TMPyP4 and overdosage of PDT.Conclusion: PARTⅠ:Different absorption peak were obtained for TMPyP4 in different solution,which means PH value and ion concentration of the solution had significant influence to the TMPyP4's absorption wavelength.Under excited the wavelength of 580nm,the maximum absorption peak occurred at the same wavelength,which means the fluorescence of the sample is weak or no fluorescence. PARTⅡ:TMPyP4-PDT has distinctive inhibition effect on Tca8113 cells,which correlated with concentration-dependent manner and energy-dependent manner.The major cell death mode is apoptosis with low concentration of TMPyP4 and mild dosage of PDT and necrosis with high concentration of TMPyP4 and overdosage of PDT.