| BackgroundNephroblastoma is a common malignant parenchymatous tumor in pediatric surgery and its prognosis is better than other tumor . From the 1990s , the rate of exairesis is almost 100% and the 5-year survival is about 80% . Early diagnosis and exairesis can obviously elevate the long term survival of patients. The recurrence of postoperation is an important factor to effect the survival. Practice has proved that if we could find and treat the recrudescent and metastatic nephroblastoma as soon as possible, part of patients could get a long-term surviving. Therefore, it is very important to follow up those post-operative patients, which is significant to early discover the recurrence of nephroblatoma treat it in time and elevate the survival. At present, monitoring the prognosis of nephroblastoma mainly relys on clinical symptom and some imageological means such as ultrasonic inspection CT Intravenous urography impact (IVP) and so on. But these inspection only could find tumors in a certain size and their sensitivities are low. In addition, nowadays we haven't found any high-specific biomarker of nephroblastoma yet. Thus, it is significant to discover a simple quick high-sensitive and high-specific method to predict the prognosis of nephroblastoma.The generation of nephroblastoma is a kinetic process referring to multi-gene and multi-factor and it is a result of oncogene activation as well as anti-oncogene inactivation. The composition and quantity of protein will change when gene mutates in cells and we can get the message of whether the tumor has been transferred or not through observing the changes of protein. The development of proteomics offers a new technological platform for the prognosis of tumor.Surface Enhanced Laser Desorption/Ionizayion Time of Flight-Mass Spectrometry (SELDI-TOF-MS) technology, a novel proteomic approach, applying the principle of genic chips and combining the laminar analysis mass-spectrum technology with protein-array, can detect many protein and polypeptide which can not be found by traditional method. SELDI-TOF-MS is an ideal means to find protein and it has rapid sensitive high-flux characteristics. We applied the surface-enhanced laser desorpton /ionization time of flight-mass spectrometry to detect the differences in biomarkers between preoperative and postoperative serums of nephroblatoma to explore a new method to monitor the prognosis.ObjectiveTo detect the differences in biomarkers between preoperative and postoperative serums of nephroblatoma to explore a new method to monitor the prognosis.Material and methods1. Clinical Material A total of 100 serum samples were from pediatric surgery of the First Affiliated hospital of Zhengzhou University: 30 preoperative nephroblastoma, 24 postoperative nephroblastoma (2 weeks after operation), 23 postoperative nephroblastoma (3 months and 6months after operation respectively ). All of nephroblastoma samples were confirmed by two pathologists. Patients consist of 16 boys and 14 girls. The range of age is from 24 days to 10 years old and the mean age is 2.8±0.1; 24 matched-pairs included 13 boys and 11 girls and the mean age is 2.6±0.1.None has received radiotherapy or chemotherapy. The control group is composed of 20 healthy children from out-patient clinic. The age and sex of healthy individuals were matched with nephroblastoma group . All the tumor blood species were all drawn at the 15d before operation and the whole blood specimens are drawn on empty stomach in the morning, and after the specimens are placed under 4℃for 1 hours, they are turned centrifugally for 10 minutes, and then the serum are extracted and preserved under -80℃.2. Main reagents and devices CHAPS, Urea, DTT, NaAC, SPA (Sinapinic acid), they are all purchased from the Pomega Company of USA. Ciphergen PBS II~+ SELDI -TOF-MS and WCX2 protein chips are purchased from Ciphergen Company of USA.3. Protein chip technical route To unfreeze the serum specimens in ice bath, turn them centrifugally at 10000 rpm under 4℃for 2 minutes. Place a 96-hole plate on the ice box, add 10 ul of U9 (9MUrea, 2%CHAPS, 1%DTT) and 5 ul of serum to each hole, vibrate at 600 rpm under 4℃for 30 minutes in cold lab chamber. Before 15 minutes of finishing the vibration, pre-process the chip. The chip is placed in the Bioprocessor, note down the chip number, add 200 ul of NaNC (100 Mm, pH4) to each hole, vibrate at 600 rpm for 2 minutes in cold lab chamber, and repeat the above-mentioned operation once. Place the 96-hole plate being processed by U9 on the ice, use a medical gun to add 185 ul of NaNC, vibrate at 600 rpm under 4℃for 2 minutes in cold lab chamber. Add 100 ul of processed specimens to the chip, place them in the cold lab chamber under 4℃combining 600 rpm for 60 minutes, swing off the remaining liquid and dry out rapidly. Add 200 ul of NaAC, after vibrating at 600 rpm for 5 minutes, swing off and dry out, repeat this operation for three times. Wash each hole twice using 200 ul of deionized water, swing off the excessive water. After the chip is air dried, each hole is added 50% saturated SPA1 ul in two stages, after drying, place them on the device for testing.4. Data collection and processing Use a protein chip whose molecular weight is known to adjust the SELDI-TOF-MS system, until the tolerance of molecular weight is less than 0.1%. Use mass spectrum reader to analyze the WCX2 protein chip combined with protein. Analysis parameters: laser strength is 170, sensitivity is 6, and the total number of each specimen collection is 140 times. The scope of data collection is 1000-30000 daltons; the optimized scope is 2000-20000 daltons. Use quality control serum to make repeated tests, the coefficients of variation of the peak value and the strength are 0.05% and 19.7% respectively.Use linear SVM classifier: The selection of feature vector uses the method of statistical filtration combining with model dependent screening, to build discrimination model, use the method of leave-one-out crossing verification to assess the discrimination result of the model.This experiment also uses discrimination analysis method to process mass spectrum data, to verify the result being processed by SVM.5. Statistical analysis After the original data of mass spectrum have been filtered out the noise and after clustering analysis, the mass-charge-ratio peaks data being preliminary screened out is carried out Wilcoxon rank sum test, the testing standard is set atα=0.01.Results1. Tumor group and normal control groupAfter the mass spectrum data of tumor group and normal control group have been preliminary filtered and screened, 352 peaks of m/z were attained. After carrying out Wilcoxon rank sum test to the relative strength, 11 m/z peaks with P value less than 0.01 were attained. From the random combination of protein peaks with remarkable variation, used SVM to screen out the combination model with the maximum youden index of the predicted value, screened out 2 markers located at 6984.5 and 6455.5, compared with the normal control group ,in tumor group, it is high expression, while in normal control group, it is low expression. Combining two potential markers as input values used the method of leave-one-out to make crossing detection, in the test collection, the specificity and sensitivity of discrimination model were 100.0% respectively.2. postoperative group and preoperative group normal control group Compared with the preoperative group, the expression of two biomarks with postoperative nephroblastoma patients were higher. Compared with the nomal control group, the expression of 19 postoperative patients with radical correction was slightly low but the difference did not reach statistic significance; the expression of 2 other cases with radical correction and 3 cases of condoning excision are slightly high but the difference does have statistics meaning (P< 0.01). Through following patients after operation (reexamine in 3 months and 6 months separately), we found that the suffers with continue high expression hadn't recidivation or metabasis ,while the patients with continue low expression didn't got radical cure or has existed potential metastasis.Conclusion1. The serum protein fingerprint model which consists of 2 pesks with m/z of 6984.5 6455.5 has perfect application value in predicting nephroblastoma prognosis.2. The combination of SELDI-TOF-MS with bioinformatics tools detects the differences in biomarkers between preoperative and postoperative serums of nephroblastoma and establishs the serum protein fingerprint model for estimating the effect of the resection of nephroblastoma.The serum protein fingerprint model offers a possible new method to predict the prognosis. |
PDF Full Text Request