| BackgroundHepatoblastoma is the most common tumor of liver in the department ofpaediatrics, holding about50%~79%of the liver tumor in paediatrics.The incidencein children is about0.5~1.5/million.The episode age is about6months~3years,with more common in boys.The pathogenesis is not clear.Thehepatoblastoma has not special symptoms in the early phase, the abdominal mass isoften found as the first symptoms,so the tumor becomes bigger when patient goes tosee a doctor.Hepatoblastoma is always single and occurs right lobe of liver.Pathological types include the fetus, the embryo and the hybrid.Imageologicalexaminations include B ultrasonic, computed tomography(and enhanced computedtomography) and hepatic arteriography. About1/2~2/3of tumor can not be removedin stage I. When the tumor was found,it has always distant metastasis as lungs. Sothe prognosis is not very good. Hepatoblastoma is a disease which can be cure.Early diagnosis and early treatment can improve the survival rate. Recently, proteomics developed quickly, according to the differences of thepurpose and method to the research, proteomics divide into structuralproteomics,Functional Proteomics and expression proteomics. The principalcorrelation techniques:two dimensional gel electrophoresis,difference gelelectrophoresise, mass spectrometry,etc.In recent years,protein chiptechnology,yeast two-hybrid system and computational analysis also use inproteomics, according to the above techniques, the variety, structure and functionof protein can make a analysis deterrmination.The proteomics applies in tumor formany aspects:1find new landmark2identify the tumor associated protein as the toolof early diagnose3explore the pathogenesis and therapy pathway of the disease.Magnetic beads protein purification can gather and pure the protein of the complexsamples in blood serum.which can improve the sensitivity in gelelectrophoresis.Surface enhanced laser desorption ionization time of flight massspectrometry (SELDI TOF MS) technology and protein chip technology give a newapproach to study protein.In the developing process of tumor,inflammatory factorconnect so intimately to it.Therefore, in the experiment, it is very important toexclude the interference of Inflammation-associated cytokines.This research mainlyuse2-DE and MS to detect the blood serum before operation,the blood serum ofSIRS patients an the blood serum of normal children, filtrate the protein markersof inflammations which is relatively specific,then to take the evaluation,and builda more advanced Liver tumor early diagnosis of serum protein fingerprint model,atlast lay the foundation for the specific proteins research of blood serum ofHepatoblastoma.According to the protein detection of tumor samples, drawfingerprint chromatogram of protein,compare the fingerprint chromatogram ofprotein with matched group,it can find out a great many different proteins,optimizing as marker combination models.Combination of many different tumormarkers improve the sensitivity and specificity of diagnosis.The technique of proteinfingerprint pattern gives new research approach and methods for filtratinglandmarks of sensitivity and specificity. ObjectiveThrough the proteomic technology, excluding the disturb of inflammationfactors,detecting the peculiar protein in serum of patients,then build a perfect serumprotein fingerprint model for early diagnosis,so it can make a foundation for the nextresearch of specific proteins in serum in hepatoblastoma.Materials and methodsMaterialsCollecting30Hepatoblastoma patients’preoperative serums from2011to2013,19Boys,and11girls, all of them are between2months and5years, their averageage is about3.5+0.1years,and the pathology after operation is proved to behepatoblastoma;Collect20SIRS patients,12boys and8girls,all of them arebetween6months and10years,their average age is about3.5+0.1years;Collect20normal children’s serums,10Boys,and10girls, all of them are between6monthsand9years, their average age is about3.8+0.2years.They are all pumped bloodspecimens to drying tube early in the morning fasting state.Put them still stand atroom temperature for1hour,centrifuge them at the speed of3000r/min for20min,then take the supernatants, mark them and save them in refrigerator at-80℃.Methods1.Serum by weak cationic magnetic beads (MB-WCX) processingPut he liver tumor patients serum, children with SIRS and the control groupserum in the ice water bath to thaw, centrifuge them at the speed of10000r/minfor5min at4℃，take5ul supernatant,apply of weak cation (MB-WCX) magneticbead chip on the sample.2. The SELDI-TOF-MS mass spectrometry system for serumprotein screeningSet the parameter, the best laser intensity, the optimum sensitivity of the massspectrometer, combine protein chip in mass spectrometer to test,record it in detail.Use SELDI-TOF-MAS,filtrate different serum proteins.Through the softwareto analyze, you can get all samples’m/z peaks,consider those whose differenceless than0.3%one category. The obtained data are chosen to carry Wilcoxonrank-sum test, In tumor group and normal group, between normal group andinflammation can get different peaks.Then can get the two groups’differentm/z peaks.The select range is+0.3%,and then found in cancer and inflammation inserum serum relatively specific protein peak.3.By adopting the method of electrophoresis for protein separationAfter the preparation of colloidal, take centrifugal serum5ml,carry theSDS-PAGE separation.Referring to the Protein Ladder,take corresponding shafts onthe film,and then mark them.4.Gel enzyme solutionRefer to the glue the enzyme solution manual,cut the film for digestion afterjoined extract,centrifuge clear liquid extract.5.Adopt MALDI-TOF-MS to make sure the the target proteinAfter use Nano HPLC in protein chip on board, identify them withMALDI-TOF/TOF.After collecting the corresponding peptides,find thecorresponding target protein in SwissProt database.Result1.Pediatric liver tumor preoperative group and normal group of thecomparison resultsAfter losing the baseline, denoising and standardized treatment of massspectrometry data of pediatric liver tumor preoperative group and normalgroup,use the clustering analysis get their peak, through Wilcoxon rank-sumtest,you can get10specific m/z peak（sP<0.01）.In pediatric liver tumor has four highexpression, high expression of six normal group,Through the SVM,filtrate youden index with the highest combination model, then get a peak at9348m/z proteinmarkers.In the pediatric liver tumor preoperative group lower expression,expression intensity were29.0+20.9(m/z9348).In normal children series highexpression, expression intensity was2036.7+881.5(m/z9348). Two groups ofcomparison difference are statistically significant (P <0.01).2.The comparative result between the children with SIRS group andcontrol groupAfter processing and statistically analyzing the mass spectrum data ofchildren with SIRS group and control group,get10specific m/z peak (P<0.01).There are6in the high expression SIRS group, and4in the lower expressiongroup.Refer combined model of the highest youden index, get a protein markerswhich peak is at5833m/z.Inflammation in children with high expression,expression intensity was1283.0+943.3(m/z5833).In normal children of significantlylower expression, expression intensity was75.7+75.1(m/z5833).3.Pediatric liver tumor specific serum peak results associated withinflammationUsing SELDI-TOF-MS platform in pediatric liver tumor group and normalgroup, inflammation group and normal group,get the specific peak; through thecomparison of two sets of results,,protein markers whose peak s are at9348m/z isfound no similar value range (+0.3%).Results show that the protein markers whosepeaks are at9348m/z are inflammatory markers.4.The identification of specific proteinsAfter the separation, purification and enzyme solution,of target protein inserum specimens, get peptides mixture,then use MALDI-TOF-TOF platform testthem. The protein whose peak is at9348m/z is identified as ApoAI. Conclusion1. M/z peak of9348proteins is identified as ApoAI, the match the detectedpeptides, amino acid sequence by sequence with the sequence in database, itsmatching rate was45%.2.Preliminary experiments show that apolipoprotein exists in other tumors, theapolipoprotein experiments provides significant reference for further research. |
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