Study On Trait Of Membrane Marker And Pre-Clinical Laboratory Diagnosis For Early Gastric Adenocarcinoma

Objective:Gastric cancer is one of the most common gastrointestinal malignancies.According to IARC first published in 2012,the global cancer statistics in the world,gastric cancer is now the third leading cause of cancer death,and therefore the diagnosis and treatment of gastric cancer research is extremely important,especially gastric adenocarcinoma.Statistics show that at the diagnosis time most patients have advanced gastric cancer,5-year survival rate after surgery is only 30%,while patients with early gastric cancer after 5-year survival rate of up to 90%,indicating that the most important way of gastric cancer treatment is early diagnosis.Currently gastric cancer diagnosis methods are endoscopy mainly,however,the endoscopy is so painful that not easily accepted by the patients,and yet there are not early gastric cancer-specific molecular markers.So it is very necessary to find new early gastric cancer-specific molecular markers,and to establish the appropriate detection methods for early diagnosis,screening and treatment.Therefore the project on the basis of previous work is to separate,extract and purify to obtain 5 new marker of early gastric cancer,to analyze chemical characterization,and to establish detection methods to reveal the clinical diagnostic value.Methods:1.Early gastric cancer cell-specific aptamer selection(1)The early gastric adenocarcinoma and normal gastric mucosa cells were isolated by 0.1%collagenase solution ? from gastric adenocarcinoma tissues.(2)The specific aptamers,bound to early gastric adenocarcinoma primary cells,were screened from a synthesized ssDNA library.During screening human gastric epithelial cells(GES)were used as counter-target cells and then their primary structure and secondary structure was predicted by DNA-sequencing and RNA-Structure software.(3)After the specificity and affinity analysis,high aptamers specificity and high affinity aptamers were obtained,as Aptamer(Ap)2.Preparation and Characterization of target membrane proteinThe target membrane proteins have been isolated by the selected 5 aptamers and biotin-streptavidin magnetic bead method.And the proteins were purified by SDS-PAGE electrophoresis and Spectrometry.The protein molecular weight,isoelectric point,subunits and other parameters were detected and analyzed by spectrometry,electrophoresis and mass spectrometry.And then the data were compared with NCBI database,determined whether they were new early gastric cancer molecular marker,and named Early gastric adenocarcinoma(EGA).3.Establishment and evaluation of new detecting markers methods(1)The establishment of detection methods:Using fluorescence-labeled aptamer recognize EGA,by fluorescence spectrophotometry,with EGA7 concentration on the fluorescence absorption values plotted to draw curve linear equation;Sensitivity analysis:preparation of a certain concentration of EGA7 solution,which was diluted into a series of concentration of the solution fluorescence photometric analysis,the lowest concentration of fluorescence absorption value is the detection sensitivity;Repeatability analysis:The intra repeat test 10 times and inter-assay test is repeated 10 times,and the upper limit of the linear range EGA7(3000ng/?l)solution as the test sample,the preparation of a series of concentrations of Ap7(?mol/l)solution,for fluorescence spectrophotometry to obtain curve,the lowest concentration value curve plateau do testing reagent concentration,to confirm scientific.(2)The detecting analysis:Using tissue cells as biological samples will be divided into early gastric cancer,advanced gastric adenocarcinoma,normal gastric mucosa were detected and analyzed using the method is to explore the diagnostic value of the markers.4.The analysis of marker from patient serumThe patient serum EGA were detected by UV spectrophotometry and identified by SDS-PAGE electrophoresis.Results:1.Tissue cells isolated by collagenase IV solution could meet the work requirements purity.22 specific aptamers of early gastric cancer cell were screened by SELEX technology for 12 rounds.Sequencing method got the primary structure and the secondary structure were obtained by RNA Structure software.After the specificity and affinity analysis,we finally get 5 high specificity:Ap1?Ap7?Ap12?Ap20?Ap26.2.The target membrane proteins(EGA1,EGA7,EGA 12,EGA20 and EGA26)have been isolated by aptamers and biotin-streptavidin magnetic bead method,The polyacrylamide gel electrophoresis and SDS-PAGE results have shown that the five target proteins are monomeric proteins.Based on the Maldi-TOF-TOF results to blast in the NCBI database,their results have shown that EGA7 is Peroxiredoxin-4,EGA 12 and EGA26 alignment results are dermcidin isoform 1 preproprotein[Homo sapiens],EGA1 is Keiatinl,was polluted by keratin.3.The ROC curve,gotten by EGA7 concentrations on fluorescence absorption,has shown that the linear equation is Y=1.6390X-912.16,(R2=0.9504);The linear range is 556.53<X<3000ng/?l,and the CV is less than 5;the sensitivity is 300ng/?l.The optimal fluorescein-labeled aptamers is 10?mol/l that is gotten from the ROC curve on fluorescence absorption and Ap7 concentrations.The SPSS analysis results have shown that there are significant difference between early gastric cancer cell group and normal gastric mucosa cell(P<0.01);significant difference between early gastric cancer cell group and progress of gastric adenocarcinoma cells(P<0.01);significant difference between the normal gastric mucosa group and advanced gastric cancer cell(P<0.01).4.The patient serum analysis has shown that five target proteins have fluorescein absorption;SDS-PAFGE showed there is cross-reactivity and the method cannot be used to detect patient serum markers,but EGA7,EGA 12 can be detected from the patient serum.Conclusion:1.The high specific aptamers have been screened for early gastric adenocarcinoma cell-SELEX technology.2.The five new membrane proteins(GA1?EGA7?EGA 12?EGA20?EGA26)are isolated and purified.And after a preliminary chemical characterization and analysis NCBI database search,their results have shown that EGA7 is Peroxiredoxin-4,EGA 12 and EGA26 alignment results are dermcidin isoform 1 preproprotein[Homo sapiens],EGA1 is Keiatinl,was polluted by keratin.Although EGA7?EGA 12 and EGA26 are proteins of known,there had not been studied as earlier gastric adenocarcinoma tumor markers,so EGA7,EGA 12 and EGA26 can be the new early gastric cancer tumor markers.3.The detecting method for EGA7 is successfully established based on aptamer C7.And there is some clinical valus.