Primary Study Of Lethal Effects By Hypocrellinb With Pdt On Human Tongue Carcinoma Tca8113Cells In Vitro

Objective: PART1: To determine the best suitable wavelength ofthe hypocrellinB(HB) dissolved in DMSO in RPMI-1640culture solutionfor the experiment. PART2：To primarily explore the lethal effects and thecells death mechanism of hypocrellinB(HB) with photodynamic therapy(PDT) on human tongue carcinoma Tca8113cells in vitro.Methods：PART1： HypocrellinB was dissolved in DMSO into Aliquid，of which the concentration was1mg/ml，and for which was onlydissolved in lipid. Then the right amount A liquid was diluted in100MLRPMI-1640culture solution with step by step into the experimentalsolution， for which the last HB concentration was0.5μM.Then theabsorption wavelength and the light absorption numerical value A wastested by BECKMAN DU series600ultraviolet and visiblespectrophotometer for the wavelength from360nm to700nm in roomtemperature. The wavelength of the largest A numerical value was chosenfor the experiment. PART2：1.The Tca8113cells were routine incubated inRPMI-1640culture solution which contained10%fetal calf serum. The cells also were incubated in water-satuerd aimosphere which contained5%CO2at37℃and were passed or frezed after3or4days.2. Thelogarithmic growing cells was chosen to blow and beat to single cellsuspension after detached，which were inoculated into96well plate of200μl for each well，of which the cells density was1×106个/ml. The chosencells were incubated for24hours. The supernate was abandoned until thecells reattached. Then the testing cells were incubated in RPMI-1640culture solution with serial concentration of HB for4hours，and then werevertical irradiated in470nm aser with different light energy. After24hours，the OD value of each well was tested by MTT process. Theproliferative inhibition rate was analyzed with certain formular.3. Thesame single cell suspension were inoculated into12well plate of1ml foreach well，which were the same cultured and PDT. Then some cells wereobserved by light microscope and fluorescence microscope，and the otherwere determined by flow cytometry (within1hour) withAnnexin-V-FITC/PI two tongue method.Result： PART1： The HB in RPMI-1640solution showed3absorption peaks separately in475nm、545nm and585nm，and theabsorption peak in475nm was obviously higher than the other two peaks.PART2：1. The cells lethal effects were not obvious in both the single HBgroup and the single PDT group.2. The cells lethal effects were obviousand different in HB-PDT group in different light density. And in definite extent, the effects were remarkable enhance in positive correlation.3. Inmiddle HB concentration and PDT laser density, the cell death mechanismwas mainly cell apoptosis.Conclusion：1：The HB in RPMI-1640solution showed the highestabsorption peak in475nm. It illustrates that the laser with nearly475nmwavelength can stimulate the largest photodynamic reaction of HB. Thuswe choose the laser equipment of470nm wavelength.2：The HB with PDThas obviously inhibition effects on human tongue carcinoma Tca8113cellsin vitro. And in definite extent, the inhibition has positive correlation withHB concentration and PDT light density.3.Although the hypocerllinB itselfhas little inhibition effect on Tca8113cells,but the inhibition effect isnotable enhanced while plus PDT, it iilustrates that the hypocrellinB hastypical photodynamic nature.And this inhibition effect is achieved byinducing cell apoptosis. Moreover, the superior cell apoptosis rate wasdetected within6h after PDT. It illustrates that the hypocrellinB has higherPDT efficiency.