Proteomics Research Of Cholangiocarcinoma

ForewordThe prognosis of cholangiocarcinoma was poor due to the difficulty of early diagnosis,little effective treatment and high-grade malignance.At present,radical surgical resection isstill the only curative option for cholangiocarcinoma.However,most patients lost theopportunity of surgical resection for the advanced disease phase after assured diagnosis.Most of them would die of biliary obstruction,biliary infection,liver failure and systemicmultiple organ failure in one year.The reason of this plight is the complexity of etiologyand pathological mechanism of cholangiocarcinoma,especially the lack of early diagnosismeans.But how do we understand the investigation the occurrence,development andevolution mechanism of cholangiocarcinoma,and explore the new and effective method ofearly diagnosis and treatment?Tumor resulted from losing control of growth and differentiation.The abnormalhyperplasy of tumor was derived from the effect of intemal factor and external factor.Canceration is a course of function transformation in tumor-related genes.The functions oftumor-related gene eventually rely on the biological functions of their correspondingprotein products.Tumor cells not only contain the same protein as normal cells,but alsocontain some specific tumor-related protein different from normal cells or lack someproteins in normal cell.During the course of DNA→mRNA→protein,there is many process and transformations,such as post-transcriptional shearing,post-translationalmodification processing and protein transfer after synthesis.The status of DNA or mRNAcannot completely represent actual level and condition of the protein.To elucidate thenature of tumor,we should comprehend the transformations of tumor-related genes,especially the dynamic change of tumor-related protein in the occurrence.If we wanted tounderstand the character of tumor,we should not only comprehend the condition oftumor-related gene,but also study dynamic transformation of tumor-related protein.Theseproteins and their fragments could enter into the circulation along with blood current andlymph stream,and the dynamic transformation of them was the reflection of physiologicalor pathological activities in tumor cell.During different disease stages,the different unique protein would be found in tumorcells.Therefore,the biomarkers in early diagnosis are expected to obtain through analyzingthe protein component in the initial stage of a disease.Serum proteome research is one ofthe most effective ways to obtain tumor markers,through obtaining a great deal of integraland dynamic serum protein spectrum.Comparative proteome is the main modes ofscreening tumor-related protein,and two-dimensional gel electrophoresis is the maintechniques of comparative proteome.Because of the poly-pathogeny and heterogeneity,the significance of a single tumormarker is limited.Tumor detection usually required a combination of multiple markers,which resulted in a new diagnostic method,serological proteomic combinational diagnosespattern.The tumor-related proteins and the combined mode determined the detectionefficiency of this diagnoses pattern.Therefore,we use comparative proteomics technologyto find the different protein pattern of cholangiocarcinoma group and control group.Thetumor-related protein with high specificity and sensitivity were selected through a series ofvalidity check.In order to obtain diagnostic information of cholangiocarcinoma rapidly,themicrofluidic chip technique were introduced into the course of tumor diagnosis.This technology has many advantages,such as less sampling,short time,miniaturization andautomation.In our previous studies controlled by Professor Zou Shengquan,we used 2-DE techniqueto separate the protein in patients' bile,who suffered from benign jaundice and malignantjaundice.Fifty-five differential proteins points were found and eight kinds of proteins weresuccessfully identified.Three kinds of proteins were closely related with the tumor.Thisstudy provided a candidate target protein for early diagnosis of cholangiocarcinoma,andvalidated the feasibility of obtaining tumor mark from bile and serum.Based on preliminary work,we continue adopt two-dimensional gel electrophoresistechnology to look for tumor-related protein from the serum of cholangiocarcinoma patients.We try to pick out the tumor markers with high diagnostic value throughclinicopathological verification experiments.The investigation provides foundation toexploit the microfluidic chip of multi-markers combined to diagnose cholangiocarcinoma.We have acquired the following conclusion.1.We establish the experiment method to separate the serum of obstructive jaundicepatient using two-dimensional gel electrophoresis technology and obtain high-quality 2-DEprotein patterns.2.We applied two-dimensional gel electrophoresis to detect the serum ofcholangiocarcinoma patients and cholangiolithiasis patients with obstructive jaundice.Twenty-five kinds of proteins were found differential expression between the two kinds ofpatients.Twenty kinds of proteins were successfully identified by mass spectrometry.Except for those same protein,eight kinds of differential expression proteins,which weretransferring,chain A of human serum transferrin,PRO2619,PRO1400,PRO2675,humanserum albumin mutant R218p complexed with thyroxine,similar to human albumin andhypothetical protein,were found low expression in the serum from the cholangiocarcinomapatients.Seven kinds of differential expression proteins,which were fibrin beta,fibrinogengamma,fibrinogen gamma chain(partial,212AA),Alpha-1-Antitrypsin,chain A of human complement component C3,chain A of T-To-Thigh quatemary transitions in humanhemoglobin and unnamed protein product,were found high expression in the serum fromthe cholangiocarcinoma patients.3.We use histochemical method to validate theexpression in tumor tissue of somecholangiocarcinoma-related protein,such as AAT,PRO2619 and TF,and discuss the rolesof these proteins during the tumorigenesis and evolution of cholangiocarcinoma.4.We examine and contrast AAT and TF using immune scattering turbidimetry incholangiocarcinoma patients' serum with some traditional tumor marker,the tumor-relatedproteins of cholangiocarcinoma screened by two-dimensional gel electrophoresis wereproved have some degree of diagnoses value. PARTⅠEstablishment of Two-Dimensional GelElectrophoresis Method Using Obstructive Jaundice Patients'SerumObjective To establish two-dimensional electrophoresis technique to screen outdifferential proteins availably from the serum of obstructive jaundice patients.Methods Three cholangiocarcinoma patients and three cholangiolithiasis patients werechose as the research object.The serums were cryopreserved after routine pretreatment.Albumin and IgG Removal Kit was applied to get rid of albumin components,bilirubin,bile salt,etc.The serums proteins were segregated using two-dimensional electrophoresistechnique and stained using coomassie brilliant blue.After staining,ImageScanner^TMgelscanner imaging system was used to obtain and preserve the map.Results We achieve serum 2-DE map with good reproducibility and highdifferentiability of cholangiocarcinoma patients and cholangiolithiasis patients withobstructive jaundice.Conclusions The serum albumin of obstructive jaundice patients could be effectivelyseparated and identified by two-dimensional gel electrophoresis-mass spectrometry.Thetechnique provides a fundamental basis for further the study to search differential proteinexpression in the serum from cholangiocarcinoma patients and cholangiolithiasis patientsand to find the biomarkers related with cholangiocarcinoma using proteomics. PARTⅡMass Spectrometry Identification of HumanCholangiocarcinoma Serum Differenital ProteinObjective To search cholangiocarcinoma-relared proteins from the serum of thepatients with obstructive jaundice using two-dimensional gel electrophoresis-massspectrometry technique.Methods We chose 10 cholangiocarcinoma patients and 10 cholangiolithiasis patientswith obstructive jaundice.The serum proteins were separated using 2-DE technique.Wescreened out the differential expressions proteins and identified through matrix-assistedlaser desorption / ionization-time of flight mass spectrometry (MALDI-TOF-TOF MS).The results of mass spectrum were analyzed through protein database.Results The serum protein 2-DE maps of cholangiocarcinoma patients andcholangiolithiasis patients were contrasted and analyzed.Twenty-five protein spots weredifferentially expressed and twenty protein spots were successfully identified by massspectrometry.Except for some same proteins,eight differential proteins,which weretransferring,chain A of human serum transferrin,PRO2619,PRO1400,PRO2675,humanserum albumin mutant R218p complexed with thyroxine,similar to human albumin andhypothetical protein,were down-regulated in cholangiocarcinoma serum.And sevendifferential proteins,which were fibrin beta,fibrinogen gamma,fibrinogen gamma chain(partial,212AA),Alpha-l-Antitrypsin,chain A of human complement component C3,chainA of T-To-Thigh quaternary transitions in human hemoglobin and unnamed protein product,were up-regulated.Conclusions Many differential proteins were found in the serum ofcholangiocarcinoma patients and cholangiolithiasis patients.Cholangiocarcinoma-relatedproteins in serum may be found by 2-DE proteomics technology.Novel proteins associatedwith cholangiocarcinoma probably included fibrin beta,fibrinogen gamma,fibrinogen gamma chain (partial,212AA),Alpha-l-Antitrypsin,chain A of human complementcomponent C3,transferring,chain A of human serum transferring and PRO2619.Thefunction and diagnoses value of these tumor-related proteins in cholangiocarcinoma needsto be validated. PARTⅢHistologic Verification Experiment of HumanCholangiocarcinoma Markers (AAT,PRO2619 and TF)Objective We validate the expression of some cholangiocarcinoma-related protein,such as AAT,PRO2619 and TF,which were selected by two-dimensional gelelectrophoresis-mass spectrometry,and discuss the roles of these proteins during thetumorigenesis and evolution of cholangiocarcinoma.Methods The expression of AAT,PRO2619 and transferrin were detected incholangiocarcinoma specimens and normal bile duct tissues specimens byimmunohistochemistry method.The results of AAT,PRO2619 and TF in the two kinds oftissues were compared.We investigated the relationship between theseproteins and theclinicopatholigic features of cholangiocarcinoma.The pertinency of these proteins was alsoanalyzed.Results1.The positive cells rates of AAT were 83.3% in cholangiocarcinoma specimens and47.4% in normal bile duct tissues specimens.There were significant difference in theexpression level of AAT between the two groups (P＜0.05).The positive cells rates ofPRO2619 were 40.5% in cholangiocarcinoma specimens and 78.9% in normal bile ducttissues specimens.There were significant difference in the expression level of PRO2619between the two groups (P＜0.05).The positive cells rates of TF were 47.6% incholangiocarcinoma specimens and 57.9% in normal bile duct tissues specimens.Therewere not significant difference in the expression level of TF between the two groups(P＞0.05).2.The expression of AAT were not correlated with gender,age,location of tumor andmetastasis of lymph node (P＞0.05),were correlated with differentiation grade,infiltrationand UICC stage of carcinoma (P＜0.05). 3.The expression of PRO2619 were not correlated with gender,age,location,metastasis of lymph node,infiltration and UICC stage of carcinoma (P＞0.05),werecorrelated with differentiation grade of carcinoma (P＜0.05).4.The expression of TF were not correlated with gender,age,location,differentiationgrade,metastasis of lymph node,infiltration and clinical UICC stage of carcinoma(P＞0.05).5.The expression of AAT,TF and PRO2619 in cholangiocarcinoma tissues had nopertinency.Conclusion1.The expression levels of AAT in cholangiocarcinoma tissues are significantly higherthan those in normal bile duct tissues,and the expression levels of PRO2619 incholangiocarcinoma tissues are significantly lower than those in normal bile duct tissues.Their expression levels are both correlated with some clinicopathological characteristics ofcholangiocarcinoma.This result suggests AAT and PRO2619 may be related to thetumorigenesis and evolution of cholangiocarcinoma,and PRO2619 especially may be anearly molecular event during tumorigenesis of cholangiocarcinoma.TF may be not involvedin tumorigenesis and evolution of cholangiocarcinoma.2.AAT,TF and PRO2619 were independent tumor-related protein during thetumorigenesis and evolution of cholangiocarcinoma. PARTⅣ.Clinical Research of Human CholangiocarcinomaMarkers (AAT and TF)Contrast with Traditional DiagnosesMethodsObjective We analyze the clinical application of AAT and TF,the candidate tumormarkers in cholangiocarcinoma patients' serum,and compare them with the traditionalserum tumor markers,such as CEA,CA19-9 as well as the imaging examination such asultrasound,CT and MRI/MRCP.We try to discuss the diagnosis value of AAT and TF incholangiocarcinoma,and find out the improvement of early diagnosis level incholangiocarcinoma through the application of integrated measure.Methods1.We examine and contrast AAT,TF,CEA and CA19-9 using immune scatteringturbidimetry in preoperative cholangiocarcinoma patients' serum with those in preoperativecholangiolithiasis patients' serum.2.We examine and contrast AAT,TF,CEA and CA19-9 using immune scatteringturbidimetry in preoperative cholangiocarcinoma patients' serum with those inpostoperative cholangiocarcinoma patients' serum.3.In order to evaluate the value of AAT and TF in cholangiocarcinoma diagnosis,wecompare the clinical diagnosis application of AAT and TF with CEA,CA19-9 and imagingdiagnosis such as ultrasound,CT,MRI / MRCP,and pathological results.Results1.The values of AAT,TF,CA19-9 and CEA in preoperative serum ofcholangiocarcinoma patients were 2.60±0.53g/L,1.98±0.48g/L,45.74±9.8ku/L,28.34±4.3μg/L,while they were 1.63±0.39g/L,1.98±0.48g/L,18.20±3.6ku/L,4.83±1.6μg/L in preoperative serum of cholangiolithiasis patients.There were significantdifference in the four tumor marker between the two groups (P＜0.05). 2.The values of AAT,TF,CA19-9 and CEA in postoperative serum ofcholangiocarcinoma patients were 1.93±0.33g/L,2.08±0.26g/L,33.27±4.2ku/L and9.83±2.2μg/L.Compared with the preoperative results,there were significant difference inAAT,CEA and CA19-9 (P＜0.05),but no significant difference in TF between the twogroups(P＞0.05).3.The sensitivity rates of using diagnostic indicator of AAT,TF,CA19-9,CEA,combined detection (four tumor markers)with parallel connection and combined detectionin series were 80%,45%,75%,45%,95% and 35%,respectively.Compare withcholangiolithiasis,the specificity rates were was 95%,70%,95%,90%,65% and 100%.The positive predictive values were 94%,60%,94%,82%,73% and 100%.The negativepredictive values were 83%,56%,79%,62%,93% and 61%.The false positive rates were5%,30%,5%,10%,35% and 0%.The diagnostic accuracies were 87.5%,72.5%,85%,67.5%,80% and 67.5%.4.The diagnostic accuracy rates of bile duct dilatation of US,CT and MRI/MRCPwere 94.4%,95% and 100%.The preoperative diagnostic accuracy rates of tumorvisualization were 66.7%,65% and 75%.The diagnostic accuracy rates of tumor locationwere 61.1%,65% and 75%,and the diagnostic accuracy rates for tumor quality were 44.4%,55% and 66.7%.Conclusion1.The examination of AAT and TF in peripheral blood is valuable to preoperativediagnosis of cholangiocarcinoma.AAT is expected to become one of diagnosis anddetection indicator of cholangiocarcinoma,which is similar to CA19-9.2.MRI/MRCP is superior to US and CT in locating the position and the nature of thetumor.It is a reliable imaging method of preoperative diagnosis in cholangiocarcinoma.3.The diagnosis step of cholangiocarcinoma can be attributed to preliminary screeningusing serological tumor markers,such as CA19-9 and AAT,combined with clinical data andusing MRI/MRCP or CT for location diagnosis. 4.Many cholangiocarcinoma-related markers could be found through proteomicstechnology.But single tumor marker possibly was difficult to improve the early diagnosisrate of cholangiocarcinoma. Two-dimensional gel electrophoresis,which is the primary technology of comparativeproteome,was taken as the main research tools in this study.Cholangiocarcinoma patientsand cholangiolithiasis patients with obstructive jaundice were taken as research object.Wetry to find out differential serum proteins of the two diseases and identify these proteinsthrough mass spectrometry.According to related literature research,α1-antitrypsin (highexpression in cholangiocarcinoma patients)and PRO2619 and transferring (high expressionin cholangiolithiasis patients)were selected to be validated the relation withcholangiocarcinoma by using immunohistochemistry histological methods.Then,theclinical diagnosis value ofα1-antitrypsin and transferrin in cholangiocarcinoma werevalidated by using scattering turbidimetry.According to the experimental results,wesummarized the conclusions as follows:1.Two-dimensional gel electrophoresis is applied in this study to separate and identifyserum proteins of the patients with obstructive jaundice.The experimental resultsdemonstrated that two-dimensional gel electrophoresis methods could effectively screen outthe differential proteins of the patients with obstructive jaundice if the samples had beenappropriate sample pretreatment.Some differential protein may be related with thepathogesis of cholangiocarcinoma and may be an early molecular event ofcholangiocarcinoma tumorigenesis.2.Some differential proteins were validated by using immunohistochemical technique.We study the function ofα1-antitrypsin,PRO2619 and transferrin in the occurrence ofcholangiocarcinoma tumorigenesis and advance.3.Some differential proteins were validated by using scattering turbidimetry technique.We compareα1-antitrypsin and transferrin with the traditional serological indicators andimaging method,and analyze them application value in the clinical diagnosis ofcholangiocarcinoma. 4.It is possible that we screen out many tumor markers from the serum ofcholangiocarcinoma patients through two-dimensional gel electrophoresis technology.Butsingle tumor marker possibly was difficult to improve the early diagnosis rate ofcholangiocarcinoma.Serological proteomic combinational diagnoses pattern andmicrofluidic chips may provide a new method for cholangiocarcinoma early diagnose.