| BackgroundNephroblastoma(Wilmstumor) is also called the embryonic tumor of kidney, is the most common retroperitoneal malignant solid tumor in children. Its incidence accounted for the first place in pediatric abdominal solid tumors, especially in children who are below the age of15occuping a large proportion in urinary reproductive system tumor. Incidence of left and right sides is almost the same, but bilateral tumor disease is less than the same time invasion, successively or simultaneously, it accounts for about3to10%of the total incidence of renal tumor. There was no difference practically in the incidence of male and female children, but there are a lot of studies have shown that the incidence of male children is slightly higher than the female children. Nowdays everyone thinks staging and classification are both important factors that affect prognosis of the tumor which has nothing to do with the size of tumor. Early diagnosis of renal tumor is of great significance to the treatment and prognosis of patient. Early symptoms of nephroblastoma in children are not so obvious, and due to its early onset age, most patients are children or infants, so this disease is easily neglected and with hospitalized time be delay. Current inspection methods are only applicable to tumor with a certain size, combined with the clinical diagnosis such as:ultrasound, discharge urinary tract imaging,computed tomography (CT) and magnetic resonance (MR), etc.; Currently the sensitivity and specificity of related diagnosis markers (including serum lactate dehydrogenase, AFP, etc.) used for the nephroblastoma is poorer, so their roles for the diagnosis or assist in diagnosis of the tumor is very limited. Above situations therefore determine the treatment emphasis (including surgery and chemotherapy drugs, etc.) should focus on the treatment of patients with advanced nephroblastoma with a explicit size, and it will inevitably affect the effect after the above-mentioned treatment. And researches in National Wilms Tumor Study (NWTS) shows that:no matter what treatments, survival rate for nephroblastoma patients will rise with their fall of installment. Therefore,Using existing experiment technology to find an simple accurate and early detection method in order to make children with nephroblastoma get earlier treatment and access to long-term survival is urgent. Ideal serum protein markers for screening and early diagnosis is of concern and research with the development of human proteomics.As early as in1994, the concept of proteomics acquired a lot of approval after it was put forward and studied. Proteome essentially refers to a study of the characteristic of the proteins. Any disease will change at the protein level, regardless of the change of quantity or quality change. This thought can build a kind of disease diagnosis system to monitor disease specificity protein level change and achieve the goal of early diagnosis, which is now an important research direction people are chasing, especially in the early diagnosis of tumors. And looking for some kind of matching specific protein of the disease has become the key issue of the research direction. With the development of the proteomics technology and with the time goes by, more and more protein biomarkers were identified. Protein markers in screening of disease and drug targets in the application of proteomics is considered one of the most effective ways, thus it can be seen, with early detection and screening,the related technologies to find accurate feasible nephroblastoma provides a possible way. Proteomics technology can rapidly give out screening pictures of tumor protein expression in different stages of gene expression,with a lot of proteins of diagnostic value in signal molecules can be found.And these proteins can provide targets for cancer treatment, provide effective tumor marker for early diagnosis. Born in2002, the surface-enhanced laser desorption/ionization time of flight-mass spectrum(SELDI-TOF-MS) protein chip technology has been widely used in many fields for searching specific biomarkers of many kinds of diseases,and has obtained a series of breakthrough. SELDI-TOF-MS technique served as a new proteomic study method, has a lot of advantages with simple operation procedures,small dosage of samples and high sensitivity, with the combination of MALDI-TOF-MS and LC/MS/MS techniques,a lot of specific associated proteins in the serum of breast, ovarian, pancreatic carcinoma have been identified.This study uses SELDI-TOF-MS technology to detect proteins of nephroblastoma before surgery group, trauma stress group and normal serum proteome, select specific protein biomarkers, and identify specific protein markers by mass spectrometry identification technology.Reject the proteins associated with traumatic stress,we can finally find something special. Depends on several important techniques in proteomics:including " SELDI-TOF-MS","MALDI-TOF-MS"," SPE"," HPLC" and " LC-LTQ-MS ",the study work can go on with the series use of above-mentioned technologies. Through preoperative serum in patients of nephroblastoma, traumatic stress protein and normal serum in children with qualitative and quantitative analysis, we find out specific proteins screened and identified in nephroblastoma serum or there are differences in content and normal children with serum protein, known as nephroblastoma specific protein markers.And the specific protein markers provides possible ways and reference for early clinical diagnosis and the treatment of nephroblastoma. In previous work, we did a lot of effort to find out a lot of relevant specific markers, however, because the tissue of carcinoma or the tissue adjacent to carcinoma tissues sometimes can produce some special nonspecific protein or material to the tumor,and make the quality and quantity of the serum proteins in the tumor be closer to some patients with inflammatory disease, trauma patients, irritable patients or patients with benign diseases. In our previous work, some bioproteins have been identified in nephroblastoma which resemble with traumatic stress disorders mentioned above, we think there may be a correlation.There are some inflammatory cytokines produced in the development of tumors because of oppression or haemorrhage.Due to interferences of traumatic stress factors with serum proteins of tumor patients on the quality and quantity, specific protein biomarkers of nephroblastoma has reduced both the credibility and applicability greatly. Root cause of this situation is that the immune system or Redox system in development of nephroblastoma in vivo process plays a key role. Therefore, in the work of screening specific protein biomarkers of nephroblastoma, we are urged to find the special proteins associated with traumatic stress or material and take it as an important job.By consulting literature, we will compare the normal serum group,nephroblastoma serum group, and trauma stress serum group, then identified proteins will be temporarily get rid of, find and remove special materials or special proteins associated with traumatic stress,confirm the specific protein biomarkers at last, and lay the foundation for the future further testing and validation.AimPreliminarily screen and identify special proteins associated with traumatic stress in nephroblastoma in children, to distinguish between tumor and trauma stress by the expression of the serum protein biomarkers, confirm the specific protein biomarkers and lay the foundation for further research on diagnosis and prognosis of nephroblastoma.MethodsBy adopting the methods of proteomics research1.1Serum sample collection and processingThe serum samples were all selected from pediatric surgery deparpment of the first affiliated hospital of Zhengzhou University, including61preoperative serum samples of nephroblastoma (type of good prognosis)(I stage28, II stage18, III stage12and IV stage3)(Stage according to the NWTS5criteria);34traumatic stress serum samples, serum collection time suffer traumatic the first1to3days);60cases from normal control healthy children. Renal tumor preoperative children, children with normal serum group and trauma subset in peripheral venous blood samples early in the morning on an empty stomach, extraction and let stand at4℃after1to2h,3000r/min, the centrifugal15min, take serum into—80℃. nephroblastoma, trauma samples were kept separate respectively with the Numbers. 1.2Serum protein screeningSELDI-TOF:tumor group and the control group of serum in the ice bath slowly melting (30～60min), after melting,4℃centrifugal10000RPM x5min, take5μL serum, add10μL U9serum processing liquid,4℃under the condition of oscillation on shaking table to mix (600rpmx30min); In Binding Buffer to U9after treatment serum dilution to200μl,4℃oscillation on shaking table to mix (600rpmx2min) then for WCX2protein chip. At first with a known molecular weight standard protein chip, All-in-One correct the SELDI mass spectrometry system error to the molecular weight<0.1%, then detect good combination of protein chips into the mass spectrometer.Original datas acquisited by Proteinchip Software3.2, laser intensity be set185, detection sensitivity7, detection limit100000m/z, optimize data collection range of2000-20000m/z. Mentioned above16kinds of inflammatory factors in each set is screening differences peak and samples for above samples for the restructuring of the protein, if differences in peak with a peak of the inflammatory factors in corresponding to the same mass-to-charge ratio, shows the differences in protein for inflammatory factor, which namely no longer carry on the purification and characterization, be more than two times compared to the special factors of traumatic meaningful peak or peak identified differences.13Data handling1) By Ciphergen Biosystems3.1correction with the original data, realizing the homogenization of the total ionic strength and the molecular weight.2) Biomarker Wizard and ZUCI protein microarray data analysis system remove the noise by discreting wavelet transform, and subtract baseline. Local extremum method to find samples in their respective peak, and filter the signal to noise ratio (S/N) which is bigger than2.The minimum threshold of Clustering analysis be set to10%, intermediate summary sample the peak of ratio (m/z) less than0.3%.3) Each of the m/z be distinguished between the significance of different peaks of sample. Do preliminary screening out of the m/z peak with Wilcoxon rank and inspect P values to measure the abilities to distinguish between different groups of each peak.4) SVM classifier to further analyze the nature of each sample spectra data, screen protein markers, look for differences in protein. Chosen in this experiment applied radial basis kernel function, y parameter be set to0.6, the penalty function be19.1.4Statistical approachMass spectrometry original data by filtering the noise, cluster analysis, t test be on different groups mass spectrometry data. Inspection standard for alpha=0.01. Determine the different proteins between groups.1.5Serum protein separationDivided through SPE technology as30%,50%,70%,100%organic phase intensity,meanwhile,carefully take notes, according to hydrophile to separate protein category.Then put it into vacuum dryer to10ul,loaded with Sample Buffer5ul,15min in boiling water,collect supernatant after centrifuged5000r/min,5minutes. According to the distance to separate different molecular weight proteins.Different molecular weight proteins in serum are separated through gel electrophoresis,cut the targeted stripes into different tubes, add in trypsin to get targeted proteins after washing, decoloration and drying.1.6MALDI-TOF-MS to determine differences in proteinBy SPE and electrophoresis separation proteins anda-Cyano-4-hydroxycinnamic Acid, take each1.5ul, mixed, put into target board, stay dry. with Cytochrome C (Molecular weight12361.96)+CHCA and Insulin+CHCA proofs. Target plate placement of MALDI instrument for testing. Tracking the target protein peak to determine protein peaks of the mass-to-charge ratio of5816serum samples.1.7Specific identification of proteinsAfter the serum protein enzyme solution liquid fill in the column on the sample; After liquid mass spectrum scanning by peptide mass fingerprint, at last use Mascot retrieval program in Swissprot database queries.1.8MALDI-TOF-MS data processing and database retrievalMass spectrometry data processing software from Launchpad2.4, PMF map retrieval using online search tools Mascot to complete. Using NCBI anthropogenic and mice protein sequence as the databases, by trypsin digestion, maximum allowable leakage cut points set to1; Fixed acetylated modification for cysteine iodine generation; Variable modifiers for methionine oxidation; The quality of the peptides tolerance being±0.3Da.Results1.SELDI detection:preoperative group and normal children group of mass spectrometry data after preliminary filtering and statistical analysis to get the P value is less than0.01m/z peak of15, from significant difference of protein peak in any combination, using SVM filter prediction of youden index (a) the difference between the positive and negative rate highest portfolio model, sieve out m/z5816protein marker, high expression in renal tumor group (intensity was2128.3±137.2) and normal children group lower expression (plus or minus30.4±7.8), the difference was statistically significant (P<0.01). By one cross method test, discriminant on the test set the two marker combination model of specificity100%, sensitivity100%.Normal group and trauma is screening the most significant difference to the m/z5816protein markers, high serum expression in trauma group (3674.6±255.7) intensity, a low expression in normal group (30.4±7.8) intensity, the difference was statistically significant (P<0.01); Renal tumor and traumatic stress group, there was no statistically significant difference between (P<0.01).2.Mass spectrometry and database retrieval:after protein samples enzymolysised, using LC-MS/MS to measure the protein peptides, enzyme solution will detect the PMF after Mascot retrieval program query protein sequence database provided by the company, check the corresponding possible protein:M/z5816which is corresponding to thioredoxin, peptides, the detected amino acid sequence by contrast with sequence in the database matching rate is20%.Conclusion1.Thioredoxin is the protein associated with traumatic stress in nephroblastoma in children.2. There is trauma stress related factor existed in the serum of nephroblastoma which identifies with non-traumatic marker apolipoprotein C-I, to a certain extent, promote the development of cancer. |
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