| Background and objective:Laboratory diagnosis of primary hepatic carcinoma(PHC)currently depends on the detection of serum alpha feta-protein(AFP).However,there are about one third of PHC patients without elevation of the serum AFP concentration,and parts of patients with benign liver diseases with elevation of serum AFP level.Therefore,founding new methods which are complementary to AFP for PHC diagnosis are very important.Nucleic acid aptamers are single-strand oligonucleotide ligands capable of binding to target molecules with high specificity and affinity,which are selected from a synthesized library of random single-stranded oligonucleotides by systematic evolution of ligands by exponential enrichment(SELEX)and considered valuable in clinical diagnostics.In our previous study,a group of aptamers were selected with PHC sera as target.In the present study,in order to create a new method based on aptamers for PHC diagnosis,we developed an approach to analyze the binding condition of an aptamer to serum based on polyacrylamide gel electrophoresis and gray measurement,by which the diagnostic values of the aptamers to PHC were evaluated.Methods:(1)Serum specimen collection:Peripheral serum specimens were collected from the patients with PHC,viral hepatitis B and cirrhosis and people for health checkup(normal control)in the First Affiliated Hospital of Nanchang University from 2009 to 2011 and refrigerated at-80℃.(2)The establishment of an approach to analyze the binding condition of an aptamer to serum based on polyacrylamide gel electrophoresis and gray measurement and detection of serum samples:Each synthetic aptamer was dissolved with SHCMK buffer solution and aliquots of gradient aptamer amounts with minimum 2pmol were electrophoresed in 12%polyacrylamide gel,stained by GelRed dye and measured by Quantity One software for the gray value of each aptamer band,and finally the optimal aptamer amount for detection was determined.A series of gradient volumes of mixed PHC serum sample with minimum 2μl were incubated with the optimal amount aptamer and electrophoresed in 12%polyacrylamide gel,stained by GelRed dye,measured by Quantity One software for the gray value of each free aptamer band,and finally the optimal serum amount for detection was determined.Based on the above results,the optimal amount of each aptamer was incubated with corresponding optimal amount serum specimen and electrophoresed and stained as described above,and then the primary gray indexes of free aptamer band of each specimen(volume,area,mean value,std.deviation and mean background)were measured and derivative gray indexes(gray ratio,signal-to-noise ratio,average net gray and variation coefficient)were calculated.(3)Data statistics and diagnostic value analyses:The significance of each primary and derivative gray index between PHC and non-PHC group was analyzed by one-way ANOVA.The diagnostic value of each gray index for PHC was evaluatedby receiver operating characteristic(ROC)curve analysis to obtain the area under the curve(AUC)and cut-off value by which the diagnostic performance(sensitivity,specificity,accuracy,predictive value and likelihood ratio)of every aptamer was calculated.Multiple-variant Logistic stepwise regression based on all gray indexes was performed in each aptamer and then the group probability of each sample was saved as a new variant(comprehensive gray index)by which a ROC curve was performed to obtain the AUC and cut-off value to calculate diagnostic performance.Multiple-variant Logistic stepwise regression based on each gray index of all aptamers was also performed and then the group probability of each sample was saved as comprehensive gray index by which a ROC curve was performed to obtain the AUC and cut-of value for calculating diagnostic performance.The results of serum AFP detection in serum samples were collected and ROC curve analysis was performed to obtain the AUC and cut-off value by which the diagnostic performance was calculated and compared with corresponding AUC and diagnostic performance of each aptamer.Results:(1)Nine aptamers were analyzed and their optimal amounts were determined as follow:5pmol for AP-HCS-9-90,AP-HCS-11-3,AP-HCS-11-6;6pmol for AP-HCS-9-10,AP-HCS-9-74,AP-HCS-11-5,AP-HCS-11-8 and AP-HCS-11-10;7pmol for AP-HCS-11-4,and the corresponding optimal serum amounts were 5μl,6μl,6μl,7μl,4μl,5μ1,5μl,6μl,7μl.(2)The case numbers of serum specimens detected were as follows:PHC 79～115,benign liver disease 90(cirrhosis,hepatitis B each 45 cases),normal control 45～63.The differences of most of gray indexes were significant among groups of PHC,benign liver disease and normal control(P<0.05-0.01),and the maximum AUC of these gray indexes for PHC diagnosis of was more than 0.9.(3)All AUCs based on probabilities of these aptamers for PHC diagnosis are more than 0.8,more detailed as follow;0.901 for AP-HCS-9-10,0.851 AP-HCS-9-74,0.988 for AP-HCS-9-90,0.891 for AP-HCS-11-3,0.902 for AP-HCS-11-4,0.916 for AP-HCS-11-5,0.939 for AP-HCS-11-6,0.899 for AP-HCS-11-8,and 0.879 for AP-HCS-11-10.(4)Multiple-variant Logistic stepwise regression based on probabilities of all aptamers showed AP-HCS-11-3,AP-HCS-11-5 and AP-HCS-11-6 in the equation,and the AUC of the probability obtained from the equation probability was 0.931.(5)Compared with AFP,the AUCs of all aptamers were larger.In the AFP negative PHC samples,the positive rates of aptamers were 78.3%～00%,and in the AFP positive benign liver disease sera,the positive rates of aptamers were 0～26.3%.Conclusions:(1)Based on the polyacrylamide gel electrophoresis and gray analysis,the approach for aptamer application in diagnosis is established and applied in detection of PHC successfully.(2)The aptamers against PHC serum are valuable in PHC diagnosis,especially based on multiple gray indexes,comparable with AFP.(3)These aptamers are valuable for the diagnosis of AFP-negative PHC and for the differentiation of AFP-positive benign liver diseases from PHC. |
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