| Background The tumor specific markers examination is the current important method for tumor diagnosis, the present examination technology including the well-rounded immunohistochemical staining and the immunofluorescence, these methods obtained the widespread approval to detection of specific markers on exsomatize tumor organization and cell. But with the deepening of cancer research, methods need to be more effective for the research of biological behavior under physiological environment, providing the most direct evidence about the tumor proliferation, invasion and metastasis; the same time, the visualization of in vivo tumor site to achieve precise positioning surgery, which is the key to improve the current diagnosis and treatment of tumor. Quantum dots (QDs), because of its unique fluorescence effects in the body can meet the requirements of target tracing in vivo. As a novel nano-probe, it can be used in many areas of biomedical fields, especially in the field of cancer research which can improve the tumor in vivo studies. However, the practical application of this emerging technology in the biological medicine is still in the initial stage, whether the labeled probe for biological applications to meet with the biological requirements of sensitivity and specificity, and whether it has good stability and feasibility of practice are still needed to explore. In this study, the QDs were used to label tumor markers in bladder urothelial carcinoma (BUC) and prostate cancer (Pca) tissues, respectively. To explore its application characteristics when compared with conventional immunohistochemistry (IHC) and fluorescein isothiocyanate labeled (FITC).Purpose Comparison of streptavidin QDs (QDs-SA 605) and IHC for detection of prostate stem cell antigen (PSCA) in BUC tissues, and comparison of secondary antibody-QDs probe (QDs-IgG 545) and FITC probe (FITC-IgG) for detection of prostate specific antigen (PSA) in Pea tissues, and on those grounds to investigate the value of QDs as a new type of fluorescent markers for biomedical applications. Methods QDs-SA 605 fluorescence labeling and IHC staining analysis of PSCA expression was performed on 96 BUC tissues, while QDs-IgG 545 and FITC-IgG fluorescence labeling analysis of PSA expression was performed on 72 Pea tissues, respectively. The intensity of staining/fluorescence were interpreted immediately after labeling under fluorescence microscope and graded on a scale of 0-3+(0, no expression; 1+, mildly positive expression; 2+, moderately positive expression; 3+, strongly positive expression). With tumor grade and stage data to compare QDs-SA 605 labeling with the IHC staining, and QDs-IgG 545 labeling with FITC-IgG labeling, analysis the similarities and differences of QDs-SA 605 and QDs-IgG 545 for detection of tumor antigen when compared with those traditional marking methods, and the consistency of them were test using Kappa analysis. Meanwhile, the attenuation of the fluorescence intensity of QDs-SA 605 and QDs-IgG 545 were observed respectively after labeled. In addition, QDs-SA 605 was also used to mark cells in vitro, and two differents QDs-SA probe (QDs-SA 605 and QDs-SA 545) with different fluorescent colors were used for bi-color labeling in BUC tissue.Results 1. The results of QDs-SA 605 labeling and IHC staining for detecting of PSCA expression in BUC tissues:Both of the QDs-SA 605 labeling and IHC staining showed a decreased rate of positive expression of PSCA accompany with the increase of BUC infiltration. In the tissue samples with tumor stage of Ta, T1, T2, T3, T4, the QDs-SA 605 labeling shows the positive expression rates were 100%,93%, 89%,83%,40%, while IHC staining were 100%,90%,89%,83%,40%. At the same time, both also showed a gradually increased intensity of PSCA expression follow with the increase of BUC pathological grade. In the pathological gradeâ… ,â…¡andâ…¢level of tissues, the QDs-SA 605 showed strong positive expression rate was 10%,21%and 58%, while the IHC staining results were 10%,21% and 50%. The consistence between QDs-SA 605 labeling and IHC staining were tested by kappa analysis and calculated K value was 0.74, z value was 11.1 (p<0.001), which illustrate the two methods were consistent in detecting the PSCA expression.2. QDs-SA cells labeling and bi-color labeling:Both QDs-SA 605 of orange-red fluorescence and QDs-SA 545 of green fluorescence were observed under simultaneous excitation, which can clealy show the expression of two different tumor markers in different location in the cell; QDs-SA 605 successful labeled the expression of PSCA in the T24 cells and clearly observed its located on the cell surface under the laser scanning confocal microscope.3. The results of QDs-IgG 545 and FITC-IgG labeling for detecting of PSA expression in Pca tissues:QDs-IgG 545 and FITC-IgG labeling both shown that the expression of PSA positive rate and intensity were decreased with the increase of Gleason score and tumor stage in Pca, the QDs-IgG 545 labeling shows the positive expression rates were 100%, 78.6% and 36%, while FITC-IgG labeling were 100%,78.6% and 32%. The changing positive rates were also observed in different tumor stages, which were 100%,69%, 62%, and 38%from T1 to T4 for QDs-IgG 545 labeling, and the similar tendency appeared in the FITC-IgG labeling that were 100%,65%,62%, and 38%. The kappa analysis calculated K value was 0.90, z value was 12.2 (p<0.001), which illustrate the QDs-IgG 545 and FITC-IgG were consistent with each other for detecting the PSA expression in Pca tissues.4. Persistent detection of fluorescence after QDs-SA 605 and QDs-IgG 545 labeling:We observed the QDs-SA 605 fluorescence continuously for 1 month and imaged it at 0,10 and 30 days. On the day of the experiment,83 of the 96 tissue samples showed detectable PSCA expression, distributed as follows:1+,2+, and 3+in 8 (8%),40 (42%), and 35 (36%) samples, respectively. Ten days after the experiment, the levels of fluorescence intensity had remained stable in most and the distribution of fluorescence intensity at 1+,2+, and 3+was 10 (10%),39 (41%), and 33 (34%) samples, respectively. Thirty days after the experiment, the distribution of fluorescence intensity at 1+,2+, and 3+was 49 (51%),29 (30%), and 0 (0%) samples, respectively. These results demonstrated that nearly 94%(78/83) of positive expression with fluorescence can last for 1 month. Unlike the FITC fluorescence under continuous illumination faded quickly and became undetectable after 6 minutes, the distribution of QDs-IgG 545 fluorescent intensity for positive PSA expression with time-varying were oserved for one month. On the day of the experiment,50 of the 72 tissue samples showed detectable PSA expression, the distribution of fluorescence intensity at 1+,2+, and 3+was 20 (27.8%),25 (34.7%) and 5 (6.9%). Ten days after the experiment, the levels of fluorescence intensity kept stable in all but 1 of the 50 samples. Twenty days after experiment, the distribution of fluorescence intensity at 1+,2+, and 3+was 27 (37.5%),23 (31.9%) and 0 (0). Thirty days after the experiment, varying degrees of fluorescence intensity decay were observed,9 of the 50 positive samples decayed to 0 and the distribution of fluorescence intensity at 1+,2+, and 3+was 38 (52.8%),3 (4.2%) and 0 (0). The rate of the fluorescence last for 1 month was 82%(41/50).Conclusion The detection performance of QDs fluorescent probe was consistent with traditional proven methods for detecting tumor-specific markers, which could take advantage of the fluorescence intensity to accurate display the tumor antigens in different expression level that correlated with tumor stage and grade in BUC and Pea, and the QDs fluorescence can maintain visible long enough to be potentially valuable for biomedical practical application. In this paper, the well-marked effect of the two types of QDs fluorescent probe in tumor tissues, and the tumor cells labeling and bi-color labeling, QDs fluorescent probe showing broad application prospects in the field of cancer research, while laid the foundation for further application.
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