| BackgroundNephroblastoma is commonly found in pediatric abdominal malignant solid tumor. First reported by Wilms,so named as Wilms’Tumor.With continuous development of pediatrics,awareness of the disease becomes more deeper.Owing to lack of early typical features,so that miss the best therapy time because of delay.In the recent years,more and more experts are committed to research early diagnosis indications of nephroblastoma,and research the typical proteins involved in the tumor through the proteomics.Through the research to get some defective genes,that have close relation with the nephroblastoma.Parts of patients children have somewhat inherited tendency. With continuous development of proteomics,to shed the light on the research about malignant tumor.SELDI-TOF-MS is most frequently applied proteomics technology in the recent years,core technology is protein chip.According to different decorations in the surface,chips contains chemistry surface chip and biology surface chip.These chips can selectively capture the proteins according to features,for example,hydrophobicity and hydrophilicity.SELDI-TOF-MS can efficiently screen related proteins in the different stage genes,especially low abundance proteins in tissues and bodily fluid closely related to tumor.it is meaningful for early diagnosis.In the recent years,researches about inflammation factors in tumor are booming so.It is manifest that different types of inflammation factors are found in primary origin and metastasis. These inflammation factors directly play role in the tumor or indirectly promote the growth of blood vessel for tumor still require further exploration.Many inflammation factors belong to protein，It will broaden the horizon that using proteomics technology research inflammation factors involved in the tumor.ObjectiveThrough the proteomics to screen inflammation factors probably existing in nephroblastoma serum. On one hand to distinguish inflammation factors and other biomarkers whose molecular weight is close to the IF in order to enhance the credibility of the results,and construct better serum protein fingerprint model for early diagnosis.On the other hand to judge whether inflammation factors indeed exist in nephroblastoma serum or not,that meaningfully guide the subsequent research between IF and growth of tumor.Materials and methodsMaterials:40preoperative nephroblastoma serums were collected from2010to2012,21male cases and19female cases,32days to8years old.The mean age is2.9±0.1years old.35postoperative serums(2weeks after operation) contain29radical operation cases and6cases condoning excision,19male cases and16female cases,from28days to8years old,The mean age is2.8±0.1.None has received radiotherapy or chemotherapy.All of nephroblastoma samples were confirmed by more than two pathologists through operation and paracentesis.The control normal group is composed of50healthy children,and inflammation group has50samples.The age and sex of healthy and inflammatory children were matched with nephroblastoma group.All the blood specimens were drawn on empty stomach in the morning,and placed under4℃for0.5-lhours.then centrifuged forl5min,3000r/min.Serums were extracted and preserved under-80℃.Methods:Treated96-hole protein chips are loaded into bioprocessor,each target is loaded with treated serum.Through editing operation program,the best scope is2000Da-20000Da,then adjust to the best laser intensity and detection sensitivity.Each chip is loaded into machine in order,through SELDI-TOF-MAS to collect results.m/z peaks in each sample are received through software,which are considered as the same class because of difference less than0.3%.m/z peaks in different groups carry out Wilcoxon rank sum test.The less P value is,the more different expression intensity.It meaningful for two different groups.So we can get typical peaks between tumor and normal group,inflammation and control group,then find out approximate value ranging about±0.3%so that get protein peak related to inflammation.Diluting tumor serum2ml with deionized water to10ml.Connecting pump and flow filter machine to collect filtrate,that is divide into10EP tube about lml each one.Mouth of tube is covered with small holes membrane,then completely frozen in the-80℃refrigerator. After it,completely dried in freezer dryer.Dilute the dried powder to250ul with deionized water.Divided through SPE technology as30%,50%,70%,100%organic phase intensity,meanwhile,carefully take notes.Then put it into vacuum dryer to10ul,loaded with Sample Buffer5ul,15min in boiling water,collect supernatant after centrifuged5000r/min,5minutes.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,drying.Collect supernatant after centrifuged10000r/min,10-15minutes.Spotting the sample on the chip,then put it into MALDI-TOF-TOF.After laser bombardment to collect peptide fragments,using Mascot software connect onto SwissProt database with comparing and matching,finally get target protein.Results1.Preoperative group and normal control groupThe original data eliminate the noise through filtration,analyzed by clustering to get peaks each group.Through Wilcoxon rank sum test,further receive15specific peaks (P<0.01).11peaks with low expression and4peaks with high expression in preoperative group.Through SVM to screen out models with maximum youden,m/z11118.8and m/z11350.8were obtained.High expression in preoperative group,intensity is1219.6±476.3(m/z11118.8) and1568.0±1043.2(m/z11350.8) Obviously low expression in normal control group,intensity is4.8±3.5and6.3±2.9.The results have statistics meaning (P<0.01)2.Inflammation group and normal control groupThe original data goes through treatment and statistics analysis,28specific peaks were obtained (P<0.01).6peaks with high expression and22peaks with low expression in inflammation group.Through SVM to screen out models with maximum youden,m/z11121.4and m/z11347.2were obtained.High expression in inflammation group.intensity is2171.4±1247.6and3282.0±3656.9.Obviously low expression in normal control group,intensity is4.8±3.5and6.3±2.9.The results have statistics meaning (P<0.01)3.Specific peaks in tumor serum about inflammationUsing SELDI-TOF-MAS to obtain specific peaks between preoperative group and normal control group,inflammation group and normal control group.From comparison to get approximate peaks ranging about±0.3%.We obtain m/z11118.8and m/z11350.8.It is proved that protein peaks related to inflammation indeed exist in tumor serum.High expression in preoperative group,obviously low expression in normal control group.Comparison with preoperative group,postoperative serums(2weeks after operation)express somewhat low.In29radical operation cases,expression intensity is4.8±4.2and6.2±4.3,The results don’t have statistics meaning (P>0.05).In6condoning excision cases,expression intensity is214.5±176.3and643.5±592.4,lower than preoperative group,The results have statistics meaning in comparison with normal control group (P<0.01)4.Identification of specific protein peaksAfter separation,purification and enzymolysis to specific protein in tumor serum,using MALDI-TOF-TOF to detect peptide fragment mixture,m/z11118.8and m/z11350.8were identified as MIF and CXCL7.Conclusion1.Two protein peaks were identified as MIF and CXCL7.It is better separate inflammation factors and other non-inflammatory serum biomarkers whose molecular weight are approximate to the identified inflammation factors,in order to construct optimized serum protein fingerprint model.It is importantly meaningful for early diagnosis,effect of operation and observation of prognosis.2.Discovery of MIF and CXCL7in nephroblastoma serum also prove that inflammation factors indeed exist in tumor serum.It does provide guidance for subsequent research between inflammation factors and growth of tumor. |
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