ABSTRACT
ObjectiveThe methods based on bladder cancer markers which could be applied to early diagnosis and postoperative recurrence monitoring of bladder cancer were current research hotspots. This study aims to screen aptamers that specifically recognize human bladder cancer cell lines (EJ, T24, BIU87) through cell-based systematic evolution of ligand by exponential enrichment (CELL-SELEX).MethodsFor CELL-SELEX screening, bladder cancer cell lines EJ, T24, and BIU87 were used as positive control cells. HCV 29 (human normal urothelial cell line), 293T (human embryonic kidney cell line), huh7 (human hepatocellular carcinoma cell line) were used as negative control cells. PCR upstream primers were labeled with FITC, downstream primer was labeled with Biotin. ssDNA fragments collected from each round were amplified by PCR, and the amplified product was then purified using a DNA purification Kit. The biotin-streptavidin magnetic separation methods were used to isolate the PCR product to obtain secondary FITC-ssDNA for the next CELL-SELEX round. The screening process was monitored by flow cytometry. ssDNA pool with the highest binding rates to bladder cancer cell lines(EJ, T24, and BIU87) was selected to PCR amplification, product purification, molecular cloning, and sequencing. According to the sequencing results, the secondary structure of the aptamer was pre-simulated by Dnaman software. Aptamer labeled with FITC was synthesized in vitro, flow cytometry was used to detect the binding rate of the aptamer to bladder cancer cell lins (EJ, T24 and BIU87).ResultsWith the advance of the CELL-SELEX process, the binding rate of FITC-ssDNA to bladder cancer cell lins (EJ, T24, and BIU87) increased gradually. By the 15th round, the binding rate of FITC-ssDNA to EJ cells reached the highest level. The apt1 had the highest enrichment among the 15th round ssDNA pool. By the 18th round, the binding rate of FITC-ssDNA to T24 or BIU87 cells reached the highest level. The apt2 and apt3 had the highest enrichment among the 18th round ssDNA pool. DNA structure prediction showed that the secondary structure of apt1, apt2, and apt3 was mainly stem-loop structure. Flow cytometry showed that the highest binding rate was FITC-apt1 to EJ cells, FITC-apt2 to T24 cells, and FITC-apt3 to BIU87 cells, respectively. There is no significant combination between these aptamers with the negative cells.ConclusionIn this study, three kinds of aptamers with high specificity for bladder cancer cell lines were successfully screened by CELL-SELEX. The apt1 can specifically recognize EJ cells, apt2 can specifically recognize T24 cells and apt3 can specifically recognize BIU87 cells, all of which provide experimental evidence for early diagnosis and targeted therapy technology research of bladder cancer.
ABSTRACT
Objective@#This study aimed to evaluate the validity and predictive value of different methods for myopia screening, and to provide the most effective method of myopia screening.@*Methods@#A total of 7 441 students of grade 1-12 selected from 36 primary and middle schools in 12 counties in Jiangsu Province were enrolled in this study for ophthalmological examination, and uncorrected visual acuity (UCVA, Plan A), noncycloplegic autorefraction (NCAR, Plan B) and combination of UCVA and NCAR (Plan C) were included in the examination. Receiver operating characteristics (ROC) curve was performed to determine the cut-off values (The gold standard is cycloplegic refraction: SE≤0.5 D), and positive predictive value, sensitivities, specificities and other indicators of three screening tests were compared and calculated.@*Results@#The AUC of the combination test for uncorrected visual acuity(UCVA) and noncycloplegic autorefraction (NCAR) was nearly 1(0.91, 95%CI=0.90-0.92), and the sensitivity, specificity, Yoden index, diagnostic coincidence rate, positive predictive value and negative predictive value were 92.4%，89.5%，0.82，91.1%，91.3%，90.8%, respectively, but the index of the younger pupils in primary schools was relatively low. The result of screening plan A for students of middle and high school students was the closest to that of myopia diagnosis.@*Conclusion@#Combination test, which was recommended by China National Health Commission, can be a practical solution for school students for myopic screening. Under the condition of no mydriatic refraction, Plan A could be considered for myopia screening especially for middle and high school students. For the younger pupils, it is suggested to use the internationally recommended myopia screening strategy, that is, SE ≤ 0.50D after mydriasis.