| 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 a very important role in the whole process. Many researchers from home and abroad confirmed the overexpression of the apoptosis related proteins, such as HSP70, HSP90, p53 and Survivin 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 HSP70, HSP90, p53 and Survivin 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 onHSP90, p53 and Survivin in human tongue cancer Tca8113 cellsMethods1.Observation of quantum dots (QD525nm,QD565nm and QD655nm) fluorescence and morphology by using confocal microscopy.2.Cell culture:selection of human tongue Tca8113 cells.3.Cell inoculation.4.Quantum dot (QD525nm,QD565nm and QD655nm) were respectively applied to tag Tca8113 strains with indirect immunofluorescence, and the expression of HSP90, p53 and Survivin in Tca8113 tongue cancer cells was observed under confocal laser microscopy.Results1. Quantum dots (QD525nm, QD565nm and QD655nm) fluorescence and morphology: QD525 was close to a circular green fluorescence, and the maximum emission wavelength was 525nm.QD655nm was close to a circular red fluorescence, and the maximum emission wavelength was 655nm. QD565nm was close to a circular orange fluorescence, and the maximum emission wavelength was 565nm.2. With laser scanning microscope QD525nm quantum dots markered HSP90 and HSP90 expression was clearly seen in human tongue cancer cell Tca8113. They was clearly seen in human tongue cancer cell cytoplasm and cell nuclear, showed red fluorescence. QD565nm quantum dots markered P53 expression was clearly seen in human tongue cancer cell Tca8113. They was clearly seen in human tongue cancer cell cytoplasm and cell nuclear, showed orange fluorescence. QD655nm quantum dots markered survivin expression was clearly seen in human tongue cancer cell Tca8113. They was clearly seen in human tongue cancer cell cytoplasm, showed red fluorescence. Chapter twoObjectsTo explore two-color immunofluorescence imaging using quantum dot with FITC,which were tagged on Survivin and HSP70 in human tongue cancer Tca8113.Then compare the photostability, specifity and strength of fluorescence signal between QD and FITC.Methods1. Cell culture:selection of human tongue Tca8113 cells.2. Cell inoculation.3. Quantum dot (QD525nm) and Fluorescein isothiocyanate (FITC) were respectively applied to tag Tca8113 strains with indirect immunofluorescence, and the expression of HSP70 in Tca8113 tongue cancer cells was observed under confocal laser microscopy. Each group was repeatedly detected for five times. We took 13 fields of vision in four directions along the hole diameter of the confocal culture dish of each sample, that is to say, each group has 65 fields of vision. The images were obtained in the same parameters. And then we used Image-Pro Plus analysis software to make quantitative analysis of each field of vision with the results output in Excel format. The sum value of Area and IOD were taken out to calculate the average fluorescence intensity of each cell. Average=IODsum/Areasum. Using software SPSS 13.0 to do the statistical analysis. Compare the specifity and strength of fluorescence signal between QD and FITC.4. QD655nm and FITC were applied to tag Tca8113 strains with two-color indirect immunofluorescence, and the expression of Survivin and HSP70 in Tca8113 tongue cancer cells was observed under confocal laser microscopy. Then the specimens were continuously illuminated with a 488nm laser for 40min 20s 391ms. The mean fluorescence intensity was measured automatically with software with the confocal microscopy. Compare the photostability between QD and FITC. ResultsQD655nm and FITC were applied to tag Tca8113:Under confocal microscope at the same time, we saw three colors fluorescence. QD655nm labeled Survivin showed red fluorescence, FITC labeled HSP70 showed green fluorescence, overlapping part of two fluorescents showed yellow fluorescence. After 40min 20s 391ms'continuous laser exposure, there was no obvious declining of QD655nm, while the FITC fluorescent signals faded very quickly and became undetectabal.Three colors fluorescence change to one color fluorescenceThe photostability, specifity and strength of fluorescence signal from QD were higher than those from fluorescein isothiocyanate (FITC).Chapter threeObjectsTo explore three-color immunofluorescence imaging using quantum dots which were tagged on HSP70, HSP90 and p53 in human tongue cancer Tca8113Methods1. Cell culture:selection of human tongue Tca8113 cells.2. Cell inoculation.3. QD655nm and QD525nm were applied to tag Tca8113 tongue cancer cells strains with two-color indirect immunofluorescence, and the expression of HSP70 and HSP90 was observed under confocal laser microscopy.4. Then QD565nm were applied to tag on p53 in human tongue cancer Tca8113 on the base of the two-color indirect immunofluorescenceResultsUnder confocal microscope at the same time, we saw five colors fluorescence. QD525nm labeled HSP70 showed green fluorescence, QD655nm labeled HSP90 showed red fluorescence, QD565nm labeled p53 showed orange fluorescence. The overlapping part of three colors fluorescent showed kermesinus fluorescence,the overlapping part of green and red fluorescent showed yellow fluorescence. Chapter fourObjectsTo investigate the expression of heat shock protein 70 (HSP70) in human squamous tongue cancer Tca8113 cells during the recovery periods of heat shock by using quantum dots-tagged fluorescence technology.Methods1. Cell culture:selection of human tongue Tca8113 cells.2. Cell inoculation.3. Heat treatment:Tca8113 cells were subjected to heat shock at 43℃for 30 min, then the cells were cultured for 2,4,6,8, and 10h respectively.4. QD525nm were applied to tag Tca8113 tongue cancer cells strains with indirect immunofluorescence, and the expression of survivin and HSP70 was observed under confocal laser microscopy.5. Quantitative analysis was performed by Image-Pro Plus (IPP).ResultsConfocal fluorescence microscopy showed that HSP70 were significantly expressed in Tca8113 cells characterized by homogeneous distribution of intensive green fluorescence. Compared with FITC tagging, quantum dots tagged fluorescence had good specificity and signal to background. At 2h after heat shock, the value began to increased significantly,4h dropped slowly, then reached the maximum value at 6h after heat shock (P<0.05),10 h after heat shock it dropped to the minimum value as much as at Oh. Conclusion1. Quantum dot marking fluoresent technology can tag HSP70, HSP90, p53 and Survivin 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 three-color immunofluorescence imaging.4.Using quantum dots as fluorescence probe can detect of the expression of heat shock protein70 in squamous tongue cancer Tca8113 cells induced by heat shock... |
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