| Background:Nephroblastoma,also called Wilms tumor,is the most common renal malignant tumor occurring in children.Since the 1990s,the surgical resection rate merely 100.0%,the overall 5-year survival rate of nephroblastoma is about 80.0%.Wilms tumor is congenital,and children also have their own characteristics can not be timely and accurate perception illness currently.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.At present,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 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.Although the in vitro cultured cell line can not reflect human internal environment and biology activity,it has many merits such as single composition,good homogen,experiment condition controlling easily,etc. Especially,when to study and research relative property,the in vitro cultured cell line can avoid some incorrect result which caused by complicated human internal environment, higher heterogeneity,etc.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 and analyse the different protein of G401,CCC-HEK-1,preoperative nephroblastoma of children and postoperative nephroblastoma of children,inquire into find new biomarkers of early detection and diagnosis of nephroblastoma,and it has rapid,sensitive,high-flux characteristics.We explored the application of these methods in preoperative diagnosis of nephroblastoma in children.Objectives:To find new biomarkers and to establish protein fingerprint models for early detection and diagnosis of nephroblastoma by SELDI-TOF-MS and cell biology.Material and methods:1.1 MaterialG401 and CCC-HEK-1 purchased from School of Basic Medicine Peking Union Medical College,A total of 100 serum samples were from pediatric surgery of the First Affiliated hospital of Zhengzhou University(30 preoperative nephroblastoma and 70 postoperative nephroblastoma).All nephroblastoma samples were confirmed by pathology.1.2 Main reagents and equipmentCHAPS,Urea,DTT,NaAC,SPA(Sinapinic acid),they are all purchased from the Pomega Company of USA.Ciphergen PBSⅡ+SELDI-TOF-MS and WCX2 protein chips are purchased from Ciphergen Company of USA.Blood serum of brepho-cattle are purchased from Hangzhou Sijiqing Biological Technology Limited Company,F12 culture solution and DMEM culture solution are purchased from School of Basic Medicine Peking Union Medical College,Trypsase are purchased from Hangzhou Jinuo Biological Technology Limited Company, RPMI-1640 are purchased from GIBCO Company,Coomassie brilliant blueG-250 are purchased from Ameresco Company of USA,Hera CO2 incubator were from GMBH Company of Germany,Inverted microscope were from Nikon Company of Japan, BP121S equi-armbalance were from Sartorious Company of Germany,Ciphergen PBSⅡ+SELDI-TOF-MS and WCX2 protein chips are purchased from Ciphergen Company of USA.1.3 Methods:1.3.1 Clinical MaterialAll the tumor blood species were all drawn at the 15 d before operation and the whole blood specimens are drawn on empty stomach in the morning,and after the specimens are placed under 4℃for one hours,they are turned centrifugally for 10 minutes,and then the serum are extracted and preserved under -86℃.preoperative nephroblastoma numbers xq1-xq30,postoperative nephroblastoma numbers xh1-xh70.1.3.2 Experiment MaterialThe in vitro cultured Wims'tumor cell line G401 was cultured by F12 solution and embryonic kidney cell line CCC-HEK-1 was cultured by DMEM solution,When they were harvested and then split by cell lysate solution,turn them centrifugally at 1000 rpm under 4℃for 5 minutes.Lose the supernatant,add in PBS,misce bene;after centrifugalization,cleaning 3 times,add in 200μl cell lysate solution, misce bene,after cell pulverizer,turn them centrifugally at 12000 rpm under 4℃for 20 minutes,abstraction supernatant.Set up G401 cell lysate solution whose concentration is 2 g/L,CCC-HEK-1 cell lysate solution whose concentration is 1g/L,and then the sample to keep by -86℃.The cell G401 harvested 3 times,The cell CCC-HEK-1 harvested 1 time,to deplete contrast of group.1.3.3 Protein chip technical routeTo unfreeze the 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 10μl of U9 (9MUrea,2%CHAPS,1%DTT) and 5μl 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 200μl 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 185μl of NaNC,vibrate at 600 rpm under 4℃for 2 minutes in cold lab chamber.Add 100μl 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 200μl 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 200μl of deionized water,swing off the excessive water.After the chip is air dried,each hole is added 50%saturated SPA1μl in two stages,after drying,place them on the device for testing.1.3.4 Data collection and processingUse 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.Every specimen is analysed by three point at least in the protein chip in order to eliminate the distinction among different points.1.3.5 Statistical analysisThe experiment collect original data automatically by using software Cliphergen Proteinchip 3.2.0 and analyse the difference of protein mass spectrum,then removed the noise and cut the base line.Auto-collected and anto-analysised the date by Biomarker Wizard software.It was defined that when S/N was lager than 5 the peak was utility.When compared two peaks,different protein was defined as the difference of the protein peaks was larger than 2 time.When compared between the serum of pateints and the protein cultured in vitro,if the mass charge ratio were same,they could be consider as the similar protein.When compared the protein cultured in vitro, they could be consider as similar protein under the condition that they had similar mass charge ratio and the difference of protein peak density was below 2 time.Date was analyzed by software SPSS 13.0.The significant difference between two groups was determined by Student's t-test.The one-way ANOVA and Student-Newman-Keuls were used to determine the significant differerces between sereral groups.In all case,P＜0.05 was considered statistically significant.Use quality control serum to make repeated tests,the coefficients of variation of the peak value and the strength are 0.05%and 15%respectively.Results:1.The protein mass spectra data of the G401 and CCC-HEK-1 cell were collected by WCX2 based on SELDI protein array technology.If the wave crest's signal/noise exceed 5,the experiment assumed it was a useable case.Then,there were 19 different wave crests which found by WCX2 between the G401 and CCC-HEK-1 cell.among those differentially expressed protein,m/z of 4287 Da,4839 Da,4984 Da,5016 Da,5322 Da,5427 Da,5930 Da,8569 Da,10095 Da,10411 Da,10844 Da,12765 Da were highly expressed,while m/z of 3020 Da,3150 Da,4083Da,4435Da,4705Da,5491Da,6881Da were lowly expressed in the G401 cell.2.The protein mass spectra data of the G401 and blood serum of preoperative nephroblastoma were collected by WCX2 based on SELDI protein array technology. If the wave crest's signal/noise exceed 5,the experiment assumed it was a useable case.Then,there were 5 similar wave crests which found by WCX2 between the G401 and blood serum of preoperative nephroblastoma.those similar expressed protein were m/z of 4633 Da,5051 Da,6143 Da,8569 Da,9295 Da.3.The protein mass spectra data of the G401 and blood serum of postoperative nephroblastoma were collected by WCX2 based on SELDI protein array technology. If the wave crest's signal/noise exceed 5,the experiment assumed it was a useable case. Then,there were 13 similar wave crests which found by WCX2 between the G401 and blood serum of postoperative nephroblastoma.those similar expressed protein were m/z of 4100 Da,4290 Da,4298 Da,4394 Da,4504 Da,4548 Da,4634 Da,4800 Da,5267 Da,5495 Da,5763 Da,8570 Da,9296 Da.Conclusion:SELDI-TOF-MS is an ideal means to find and analyse the different protein of G401,CCC-HEK-1,preoperative nephroblastoma of children and postoperative nephroblastoma of children,inquire into find new biomarkers of early detection and diagnosis of nephroblastoma,and it has rapid,sensitive,high-flux characteristics.We explored the application of these methods in preoperative diagnosis of nephroblastoma in children. |
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