Application Of Quantum Dot Fluorescence In Labeling Bcl-2, Bax In Oral Squamous Cell Carcinomas

Cancer is one of the most important diseases influence the human badly.Oral cancer is the malignant tumor often occurred on the pate.The development of the oral cancer is a process with multiple factors and stages.Many studies have evidenced apoptosis of the cells play an very important role in the whole process.Many researchers from home and abroad confirmed the overexpression of the apoptosis related proteins,such as bcl-2,bax in oral squamous cancers.The organic dyes have been used in labeling the biomacromolecule for a very long time,which was to study the interaction between various proteins and cells function.However,because of the shortcomings of organic dyes,such as narrow excitation bands,low photostability, Short fluorescence lifetimes,the applications as markers in many areas were limited. Recently,nanoparticles fluorescent probe based on the semiconductor quantum dots (Quantum dot,QD) is widely observed because of its unique optical properties.QD technology has a very promising prospect in the imagery of the biomedical field of molecular,cellular and endosomatic imaging,particularly in the study of biological imaging of oncology.Studies on the Bcl-2,bax protein of oral squamous cancer cells with quantum dots have not been reported.Chapter oneObjectsTo evaluate the application of quantum dots(QD) fluorescence labeling technique on bcl-2,bax in human tongue cancer Tca8113 cells,and to compare the light stability with the traditional fluorescein isothiocyanate(FITC) method.Methods1.Observation of quantum dots(QD605 and QD545 ) fluorescence and morphology by using confocal microscopy.2.Cell culture:selection of human tongue Tca8113 cells.3.Quantum dots(QD605) and QD545 were respectively applied to tag Tca8113 strains with indirect immunofluorescence,and the expression of bcl-2,bax in Tca8113 tongue cancer cells was observed under confocal laser microscopy.4.Fluorescein isothiocyanate(FITC) was applied to tag Tca8113 strains with indirect immunofluorescence,and the expression of bcl-2 in Tca8113 tongue cancer cells was observed under confocal laser microscopy.The fluorescent signal strength was also compared between those from OD605 labeling and FITC.5.Photostability comparison between QD605 and FITC. Confocal microscope observation and parameter setting:compare the photostability of QD and FITC,the specimens were continuously illuminated with a 488nm laser (QD605) and a 456nm laser(QD545)(Ar laser power 100mW ) for 1h.QD605 used emission filters from 590nm to 615nm,and QD545 used emission filters from 505nm to 535nm.FITC used emission filters from 505nm to 535um.The mean fluorescence intensity was measured automatically with software with the confocal microscopy.Results1.Quantum dots(QD605 and QD545 ) fluorescence and morphology:QD605 was close to a circular red fluorescence,and the maximum emission wavelength was 605nm.QD545 was close to a circular green fluorescence,and the maximum emission wavelength was 545nm.2.With laser scanning microscope QD605 quantum dots markered bcl-2 expression was clearly seen in human tongue cancer cell Tca8113.They was clearly seen in human tongue cancer cell cytoplasm,nuclear membrane,and Showed red fluorescence.With laser scanning microscope QD545 quantum dots markered bax expression was clearly seen in human tongue cancer cell Tca8113.They was clearly seen in human tongue cancer cell cytoplasm,and Showed red fluorescence.3.FITC-specific markered bcl-2 expression was clearly seen in human tongue cancer cell cytoplasm,and the expression was not clearly seen in the nuclear membrane.They showed green fluorescence.4.The specifity and strength of fluorescence signal were higher than those from fluorescein isothiocyanate(FITC).After one hour's continuous laser exposure, there was no obvious declining of fluorescent markers,while the fluorescein isothiocyanate fluorescent signals faded very quickly and became undetectabal in 1h.Chapter twoObjectsTo explore two-color immunofluorescence imaging using quantum dots in bcl-2,bax protein of oral squamous cells.Methods1.Cell culture:selection of human tongue Tca8113 cells.2.Quantum dots(QD605) and Quantum dots(QD545) were applied to tag Tca8113 strains with two-color indirect immunofluorescence,and the expression of bcl-2, bax in Tea8113 tongue cancer cells was observed under confocal laser microscopy.Confocal microscope observation and parameter setting were the same as chapter one.ResultsQuantum dots markered bcl-2,bax were specific detected in the tongue cancer by under confocal microscope at the same time:bcl-2,bax protein are overexpressed in tongue cancer cells.Three-color fluorescence was seen in human tongue cancer cell Tca8113,and QD605 labeled bcl-2 showed red fluorescence,QD545 labeled bax showed green fluorescence,overlapping part of two fluorescent showed yellow fluorescence.after one hour's continuous laser exposure,there was no obvious declining of fluorescent markers.Chapter threeObjectsTo explore the influence of quantum dots on the oral squamous living cells,and tracer bcl-2,bax protein in oral squamous living cells.Methods1.The influence by quantum dots in oral squamous living cells1.1 Cell culture1.2 Experimental Grouping:cells were divided into control and four experimental groups(QDS:20nmol/L,30nmol/L,40nmol/L,50nmol/L),each group set six parallel-hole.1.3 Observation and and analysis:different concentrations of QD synchronization added to the experimental well in the dose group instantaneously,37℃,5%CO2 incubator cultivate.Take out of the 96well(culture plate) after 4,8,12,16,20h of the experiment.20μl MTT solution was added(20μl / well)in each hole,and cells were then incubated for 4h at 37℃,5%CO2.Medium was removed,and the cells were lysed with dimethylsulfoxide(DMSO,150μl / well),then the plate was vibrated for 10min to dissolve the crystal at room temperature,and absorbance was measured at 490nm using microplate reader.1.4 Statistical analysis:statistical analysis was performed by using spss13.0 statistical software,The significance level was set at p＜0.05.The results of statistical analysis were measured,and the cell growth curve were determined by MTT assay in a blank control group and different concentrations QD group.2.Observing of bcl-2 protein in oral squamous living cells with using quantum dots2.1 Cell culture2.2 Cell Climb tablets2.3 Tongue Tca8113 living cells bcl-2 protein and quantum dots were incubated 0.5h,1.0h,1.5h and 2.0h,and the fluorescence imaging was observed under confocal laser microscopy. 2.4 Confocal microscope observation and parameter setting:the specimens were continuously illuminated with a 488nm laser(Ar laser power 100mW),and QD605 used emission filters from 590nm to 615nm.3.Observing of bax protein in oral squamous living cells with using quantum dots3.1 Cell culture3.2 Cell Climb tablets3.3 Tongue Tca8113 living cells bax protein and quantum dots were incubated 0.5h, 1.0h.1.5h and 2.0h,and the fluorescence imaging was observed under confocal laser microscopy.3.4 Confocal microscope observation and parameter setting:the specimens were continuously illuminated with a 456nm laser(Ar laser power 100mW),and QD605 used emission filters from 535nm to 555nm.Results1.The results showed that each experimental group and control group was not significant at 4 hours,8 hours(P＞0.05);Quantum dots final concentration of 20nmol/L group and blank control group were not statistical significant at 12 hours, 16 hours,20 hours(P＞0.05),and the other group and blank control group were statistically significant(P＜0.05).2.The bcl-2 protein observing in oral squamous living cells by quantum dots(QD605) showed:at 0.5h,QD605 and bcl-2 protein bond,and red fluorescence was mainly distributed in cytoplasm of the edge,close to the nuclear membrane has a mass of red fluorescence.At 1.0 h,the red fluorescence distributed in the cytoplasm,the distribution of more casual,and there is scattered and gathered at the red fluorescent area;At 1.5h,the red fluorescence distributed in the cytoplasm,the red fluorescence was more clearly;At 2.0 h,the red fluorescence is not only distributed in the cytoplasm,but also reached the nuclear membrane,where red fluorescence become more pronounced.3.The bax protein observing in oral squamous living cells by quantum dots(QD545) showed:at 0.5h,QD545 and bax protein bond,green fluorescence was mainly distributed in the cytoplasm,the distribution of more casual;At 1.0 h,Green fluorescence distributed in the cytoplasm,and green fluorescence distributed concentration than 0.5 hours:At 1.5h.green fluorescence distributed in throughout the cytoplasm,and distributed evenly;At 2.0 h.in addition to the distribution of green fluorescence in the cytoplasm,some green fluorescence has been the distribution of the nuclear membrane.Conclusion1.Quantum dot marking fluoresent technology can tag bcl-2,bax in cancer cells.2.Quantum dot have much higher photostability and longer fluorescence lifetimes than the traditional organic dye.3.Quantum dots can simultaneously lable two-color immunofluorescence imaging in bcl-2,bax protein of oral squamous cells.4.Concentration of quantum dots have no significant influence on growing of cell less than 20nmol/L.5.Observing of protein in oral squamous living cells with quantum dots.