胆管癌蛋白组学研究
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摘要
第一部分梗阻性黄疸患者血清样本双向凝胶电泳技术的建立
目的建立能够有效筛选梗阻性黄疸患者血清样本中差异蛋白的双向凝胶电泳技术。
方法选择有梗阻性黄疸的胆管癌和胆管结石患者各3例，收集空腹血清，进行常规预处理后冷冻保存，应用Albumin and IgG Removal Kit去除血清中高丰度蛋白、胆红素和胆盐等成份，双向凝胶电泳技术分离血清蛋白样本，蛋白分离凝胶采用考马斯亮蓝染色，ImageScanner~(TM)凝胶扫描仪成像系统获取并保存图像。
结果获得了合并梗阻性黄疸的胆管癌和胆管结石患者的重复性好、分辨率高的血清蛋白样本双向凝胶电泳图谱。
结论双向凝胶电泳-质谱技术能够有效地分离和鉴定梗阻性黄疸患者的血清蛋白，这项技术为对比分析胆管癌和胆管结石患者血清蛋白的差异、筛选胆管癌特异性肿瘤标志物提供了方法学基础。
第二部分人胆管癌血清差异蛋白的质谱鉴定
目的通过双向凝胶电泳-质谱技术寻找梗阻性黄疸患者血清中的胆管癌相关蛋白。
方法选择有梗阻性黄疸的胆管癌和胆管结石患者各10例，应用双向凝胶电泳技术-质谱技术分离其血清蛋白样本，筛选出差异表达的蛋白点，应用基质辅助激光解析电离飞行时间串联质谱技术进行质谱鉴定，并通过蛋白质数据库查询质谱鉴定结果，获得差异蛋白的详细信息。
结果对比分析胆管癌和胆管结石患者血清蛋白的双向凝胶电泳分离图谱，发现25个差异蛋白质点并选取进行质谱鉴定，其中20个差异蛋白质点获得成功鉴定，鉴定成功率80％；去除相同重复蛋白质后，有8种差异蛋白在胆管结石患者血清中高表达，分别为转铁蛋白、转铁蛋白A链、PRO2619、PRO1400、PRO2675、人血清白蛋白变构体、人血清白蛋白类似物和假设蛋白，有7种差异蛋白在胆管癌患者血清中高表达，分别为纤维蛋白β、纤维蛋白原γ、纤维蛋白原γ片段、α1-抗胰蛋白酶、补体C3的A链、人血红蛋白A链和未命名蛋白产物。
结论胆管癌与胆管结石患者的血清蛋白中存在较多差异表达的蛋白质，利用双向凝胶电泳技术有望筛选出与胆管癌相关的肿瘤标志物。在筛选出的差异蛋白中可能与胆管癌相关的有纤维蛋白β、纤维蛋白原γ、纤维蛋白原γ片段、α1-抗胰蛋白酶、补体C3的A链、转铁蛋白、转铁蛋白A链和PRO2619，它们在胆管癌发生与演进中所起的作用以及在胆管癌诊断中的价值尚有待进一步验证。
第三部分人胆管癌血清标志物AAT、PRO2619和TF的组织学验证实验
目的检测双向凝胶电泳-质谱技术筛选出的部分胆管癌相关蛋白α_1-抗胰蛋白酶(α1-antitrypsin，AAT)、PRO2619和转铁蛋白(transferrin，TF)在胆管癌组织中的表达，分析AAT、PRO2619和TF的表达与胆管癌临床病理特性的关系，探讨它们在胆管癌发生和演进过程中的作用。
方法用免疫组织化学方法检测AAT、PRO2619和TF在胆管癌组织和正常胆管组织石蜡标本切片中的表达水平，分析它们与胆管癌临床病理特性关系，以及三种蛋白在胆管癌组织中表达情况是否具有相关性。
结果
1．AAT在胆管癌组织中的阳性表达率为83．3％，在正常胆管组织中的阳性表达率为47．4％，二者差异有统计学意义P＜0．05)；PRO2619在胆管癌组织中的阳性表达率为40．5％，在正常胆管组织中的阳性表达率为78．9％，二者差异有统计学意义(P＜0．05)；TF在胆管癌组织中的阳性表达率为47．6％，在正常胆管组织中的阳性表达率为57．9％，二者差异无统计学意义(P＞0．05)。
2．AAT在胆管癌组织中的表达水平与患者的性别、年龄、肿瘤部位、淋巴结转移无明显关系(P＞0．05)，与癌组织分化程度、周围浸润和UICC分期有明显关系(P＜0．05)。
3．PRO2619在胆管癌组织中的表达水平与患者的性别、年龄、肿瘤部位、淋巴结转移、周围浸润及UICC分期均无明显关系(P＞0．05)，与癌组织分化程度有明显关系(P＜0．05)；
4．TF蛋白表达水平与患者的性别、年龄、肿瘤部位、分化程度、淋巴结转移、周围浸润和UICC分期均无明显关系(P＞0．05)。
5．AAT、TF、PRO2619在胆管癌组织中的表达情况之间无相关性(P＞0．05)。
结论
1．AAT在胆管癌组织中表达增高、PRO2619在胆管癌组织中表达降低，并与胆管癌某些临床病理特性存在明显关系，说明AAT和PRO2619可能与了胆管癌的发生和恶性演进过程有关，特别是PRO2619的变化，有可能是胆管癌发生早期的分子改变。TF可能与胆管癌发生、发展和演进过程的关系不大。
2．在胆管癌发生、发展和演进过程中，AAT、TF和PRO2619所发挥的作用可能不具有相关性。
第四部分人胆管癌血清肿瘤标志物AAT、TF与传统诊断方法的临床对比研究
目的分析本研究所筛选的胆管癌血清肿瘤标志物AAT、TF的血清学验证情况，并与传统血清肿瘤标志物CEA、CA19-9以及超声、CT和MRI／MRCP等影像学检查进行对比，探讨检测血清中AAT、TF在胆管癌诊断中的价值，以及如何通过应用综合手段提高胆管癌早期诊断水平。
方法
1．应用免疫散射比浊法检测胆管癌和胆管结石患者术前血清中AAT、TF水平以及CEA、CA19-9水平，对比两组之间的差异。
2．应用免疫散射比浊法检测胆管癌患者术前和根治性切除术后1个月时血清中AAT、TF水平以及CEA、CA19-9水平，对比术前、术后的差异。
3．将AAT、TF在胆管癌中的诊断应用情况与CEA、CA19-9以及超声、CT、MRI／MRCP等影像学诊断检查、术后病理结果进行对比分析，初步评判AAT、TF在胆管癌诊断中的临床应用价值。
结果
1．胆管癌患者术前血清中AAT、TF、CA19-9、CEA含量分别为2．60±0．53 g／L、1．98±0．48 g／L、45．74±9．8 ku／L、28．34±4．3μg/L，而胆管结石患者术前血清中AAT、TF、CA19-9、CEA含量分别为1．63±0．39 g／L、1．98±0．48 g／L、18．20±3．6 ku／L、4．83±1．6μg／L，两组间四种指标的差异均具有统计学意义(P＜0．05)。
2．胆管癌患者根治性术后血清中AAT、TF、CA19-9、CEA含量分别为1．93±0．33g／L、2．08±0．26 g／L、33．27±4．2 ku／L、9．83±2．2μg／L，与术前结果相比，AAT、CEA、CA19-9三项指标存在显著性差异，具有统计学意义(P＜0．05)，而TF值手术前后的差异不具有统计学意义(P＞0．05)。
3．AAT、TF、CA19-9、CEA、四项指标联合并联检测和四项指标联合串联检测对胆管癌诊断的敏感性分别为80％、45％、75％、45％、95％和35％；以胆管结石患者为对照，诊断特异性分别为95％、70％、95％、90％、65％和100％；阳性预测值94％、60％、94％、82％、73％和100％；阴性预测值83％、56％、79％、62％、93％和61％；假阳性率5％、30％、5％、10％、35％和0％；诊断准确度分别为87．5％、72．5％、85％、67．5％、80％和67．5％。
4．各种影像学检查中，US、CT、MRI／MRCP的肝内外胆管扩张发现率分别为94．4％、95％、100％，肿块显示率分别为66．7％、65％、75％，定位诊断准确率分别为61．1％、65％、75％，定性诊断准确率分别为44．4％、55％、66．7％。
结论
1．检测外周血清中AAT、TF的值可能对胆管癌的诊断具有一定价值，其中AAT在胆管癌诊断与监测的价值可能与CA19-9近似。
2．MRI／MRCP对胆管癌定位和定性诊断优于US、CT，是胆管癌术前可靠的影像学诊断手段。
3．目前胆管癌的诊断步骤可归结为，血清学检测CA19-9、AAT等肿瘤标志物结合临床资料进行初步筛选，然后采用MRI/MRCP或CT进行定位诊断。
4．通过蛋白质组学技术可以发现较多胆管癌血清肿瘤标志物，但目前筛选出的任何单一胆管癌标志物可能均难以提高胆管癌的早期诊断率。
小结
本课题从比较蛋白质组学的主要方法——双向凝胶电泳技术着手，以合并梗阻性黄疸的胆管癌和胆管结石患者为研究对象，筛选出两种疾病血清中的差异蛋白质，并进行质谱鉴定。根据文献研究结果，选择在胆管癌患者血清表达增强的α_1-抗胰蛋白酶和胆管结石患者血清中表达增强的PRO2619、转铁蛋白，应用免疫组织化学方法进行组织学验证，分析它们与胆管癌发生和演进的关系；然后选择α_1-抗胰蛋白酶和转铁蛋白进行血清学验证，分析它们在胆管癌诊断中的应用价值。本研究获得以下结论：
1．本课题以梗阻性黄疸患者的血清为研究对象，应用双向凝胶电泳技术分离并鉴定差异表达的血清蛋白。结果显示，通过相应的标本预处理技术，双向凝胶电泳方法可以有效的筛选出梗阻性黄疸患者血清中的差异蛋白，其中有些蛋白可能与胆管癌的发生发展有关，且有可能是胆管癌发生中的早期分子事件。
2．通过免疫组织化学技术，对筛选出的部分差异蛋白进行了组织学验证，分析α_1-抗胰蛋白酶、PRO2619和转铁蛋白在胆管癌发生发展中可能所起的作用。
3．通过免疫散射比浊法对筛选出的部分差异蛋白进行了血清学验证，并与传统血清学指标和影像学方法进行对比，分析α_1-抗胰蛋白酶和转铁蛋白在胆管癌临床诊断中的应用价值。
4．通过双向凝胶电泳-质谱技术，我们可以从胆管癌患者的血清中筛选出较多的肿瘤标志物，但目前筛选出的任何单一胆管癌标志物尚难以提高胆管癌的早期诊断率；血清蛋白质组合诊断模式和联合应用这些肿瘤标志物开发出微流控检测芯片，可能是未来提高胆管癌早期诊断率的新途径。
Foreword
The 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'
Serum
Objective 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~(TM)gelscanner 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 Protein
Objective 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.
Results
1.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.
Conclusion
1.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 Diagnoses
Methods
Objective 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.
Methods
1.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.
Results
1.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%.
Conclusion
1.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.
目的建立能够有效筛选梗阻性黄疸患者血清样本中差异蛋白的双向凝胶电泳技术。
方法选择有梗阻性黄疸的胆管癌和胆管结石患者各3例，收集空腹血清，进行常规预处理后冷冻保存，应用Albumin and IgG Removal Kit去除血清中高丰度蛋白、胆红素和胆盐等成份，双向凝胶电泳技术分离血清蛋白样本，蛋白分离凝胶采用考马斯亮蓝染色，ImageScanner~(TM)凝胶扫描仪成像系统获取并保存图像。
结果获得了合并梗阻性黄疸的胆管癌和胆管结石患者的重复性好、分辨率高的血清蛋白样本双向凝胶电泳图谱。
结论双向凝胶电泳-质谱技术能够有效地分离和鉴定梗阻性黄疸患者的血清蛋白，这项技术为对比分析胆管癌和胆管结石患者血清蛋白的差异、筛选胆管癌特异性肿瘤标志物提供了方法学基础。
第二部分人胆管癌血清差异蛋白的质谱鉴定
目的通过双向凝胶电泳-质谱技术寻找梗阻性黄疸患者血清中的胆管癌相关蛋白。
方法选择有梗阻性黄疸的胆管癌和胆管结石患者各10例，应用双向凝胶电泳技术-质谱技术分离其血清蛋白样本，筛选出差异表达的蛋白点，应用基质辅助激光解析电离飞行时间串联质谱技术进行质谱鉴定，并通过蛋白质数据库查询质谱鉴定结果，获得差异蛋白的详细信息。
结果对比分析胆管癌和胆管结石患者血清蛋白的双向凝胶电泳分离图谱，发现25个差异蛋白质点并选取进行质谱鉴定，其中20个差异蛋白质点获得成功鉴定，鉴定成功率80％；去除相同重复蛋白质后，有8种差异蛋白在胆管结石患者血清中高表达，分别为转铁蛋白、转铁蛋白A链、PRO2619、PRO1400、PRO2675、人血清白蛋白变构体、人血清白蛋白类似物和假设蛋白，有7种差异蛋白在胆管癌患者血清中高表达，分别为纤维蛋白β、纤维蛋白原γ、纤维蛋白原γ片段、α1-抗胰蛋白酶、补体C3的A链、人血红蛋白A链和未命名蛋白产物。
结论胆管癌与胆管结石患者的血清蛋白中存在较多差异表达的蛋白质，利用双向凝胶电泳技术有望筛选出与胆管癌相关的肿瘤标志物。在筛选出的差异蛋白中可能与胆管癌相关的有纤维蛋白β、纤维蛋白原γ、纤维蛋白原γ片段、α1-抗胰蛋白酶、补体C3的A链、转铁蛋白、转铁蛋白A链和PRO2619，它们在胆管癌发生与演进中所起的作用以及在胆管癌诊断中的价值尚有待进一步验证。
第三部分人胆管癌血清标志物AAT、PRO2619和TF的组织学验证实验
目的检测双向凝胶电泳-质谱技术筛选出的部分胆管癌相关蛋白α_1-抗胰蛋白酶(α1-antitrypsin，AAT)、PRO2619和转铁蛋白(transferrin，TF)在胆管癌组织中的表达，分析AAT、PRO2619和TF的表达与胆管癌临床病理特性的关系，探讨它们在胆管癌发生和演进过程中的作用。
方法用免疫组织化学方法检测AAT、PRO2619和TF在胆管癌组织和正常胆管组织石蜡标本切片中的表达水平，分析它们与胆管癌临床病理特性关系，以及三种蛋白在胆管癌组织中表达情况是否具有相关性。
结果
1．AAT在胆管癌组织中的阳性表达率为83．3％，在正常胆管组织中的阳性表达率为47．4％，二者差异有统计学意义P＜0．05)；PRO2619在胆管癌组织中的阳性表达率为40．5％，在正常胆管组织中的阳性表达率为78．9％，二者差异有统计学意义(P＜0．05)；TF在胆管癌组织中的阳性表达率为47．6％，在正常胆管组织中的阳性表达率为57．9％，二者差异无统计学意义(P＞0．05)。
2．AAT在胆管癌组织中的表达水平与患者的性别、年龄、肿瘤部位、淋巴结转移无明显关系(P＞0．05)，与癌组织分化程度、周围浸润和UICC分期有明显关系(P＜0．05)。
3．PRO2619在胆管癌组织中的表达水平与患者的性别、年龄、肿瘤部位、淋巴结转移、周围浸润及UICC分期均无明显关系(P＞0．05)，与癌组织分化程度有明显关系(P＜0．05)；
4．TF蛋白表达水平与患者的性别、年龄、肿瘤部位、分化程度、淋巴结转移、周围浸润和UICC分期均无明显关系(P＞0．05)。
5．AAT、TF、PRO2619在胆管癌组织中的表达情况之间无相关性(P＞0．05)。
结论
1．AAT在胆管癌组织中表达增高、PRO2619在胆管癌组织中表达降低，并与胆管癌某些临床病理特性存在明显关系，说明AAT和PRO2619可能与了胆管癌的发生和恶性演进过程有关，特别是PRO2619的变化，有可能是胆管癌发生早期的分子改变。TF可能与胆管癌发生、发展和演进过程的关系不大。
2．在胆管癌发生、发展和演进过程中，AAT、TF和PRO2619所发挥的作用可能不具有相关性。
第四部分人胆管癌血清肿瘤标志物AAT、TF与传统诊断方法的临床对比研究
目的分析本研究所筛选的胆管癌血清肿瘤标志物AAT、TF的血清学验证情况，并与传统血清肿瘤标志物CEA、CA19-9以及超声、CT和MRI／MRCP等影像学检查进行对比，探讨检测血清中AAT、TF在胆管癌诊断中的价值，以及如何通过应用综合手段提高胆管癌早期诊断水平。
方法
1．应用免疫散射比浊法检测胆管癌和胆管结石患者术前血清中AAT、TF水平以及CEA、CA19-9水平，对比两组之间的差异。
2．应用免疫散射比浊法检测胆管癌患者术前和根治性切除术后1个月时血清中AAT、TF水平以及CEA、CA19-9水平，对比术前、术后的差异。
3．将AAT、TF在胆管癌中的诊断应用情况与CEA、CA19-9以及超声、CT、MRI／MRCP等影像学诊断检查、术后病理结果进行对比分析，初步评判AAT、TF在胆管癌诊断中的临床应用价值。
结果
1．胆管癌患者术前血清中AAT、TF、CA19-9、CEA含量分别为2．60±0．53 g／L、1．98±0．48 g／L、45．74±9．8 ku／L、28．34±4．3μg/L，而胆管结石患者术前血清中AAT、TF、CA19-9、CEA含量分别为1．63±0．39 g／L、1．98±0．48 g／L、18．20±3．6 ku／L、4．83±1．6μg／L，两组间四种指标的差异均具有统计学意义(P＜0．05)。
2．胆管癌患者根治性术后血清中AAT、TF、CA19-9、CEA含量分别为1．93±0．33g／L、2．08±0．26 g／L、33．27±4．2 ku／L、9．83±2．2μg／L，与术前结果相比，AAT、CEA、CA19-9三项指标存在显著性差异，具有统计学意义(P＜0．05)，而TF值手术前后的差异不具有统计学意义(P＞0．05)。
3．AAT、TF、CA19-9、CEA、四项指标联合并联检测和四项指标联合串联检测对胆管癌诊断的敏感性分别为80％、45％、75％、45％、95％和35％；以胆管结石患者为对照，诊断特异性分别为95％、70％、95％、90％、65％和100％；阳性预测值94％、60％、94％、82％、73％和100％；阴性预测值83％、56％、79％、62％、93％和61％；假阳性率5％、30％、5％、10％、35％和0％；诊断准确度分别为87．5％、72．5％、85％、67．5％、80％和67．5％。
4．各种影像学检查中，US、CT、MRI／MRCP的肝内外胆管扩张发现率分别为94．4％、95％、100％，肿块显示率分别为66．7％、65％、75％，定位诊断准确率分别为61．1％、65％、75％，定性诊断准确率分别为44．4％、55％、66．7％。
结论
1．检测外周血清中AAT、TF的值可能对胆管癌的诊断具有一定价值，其中AAT在胆管癌诊断与监测的价值可能与CA19-9近似。
2．MRI／MRCP对胆管癌定位和定性诊断优于US、CT，是胆管癌术前可靠的影像学诊断手段。
3．目前胆管癌的诊断步骤可归结为，血清学检测CA19-9、AAT等肿瘤标志物结合临床资料进行初步筛选，然后采用MRI/MRCP或CT进行定位诊断。
4．通过蛋白质组学技术可以发现较多胆管癌血清肿瘤标志物，但目前筛选出的任何单一胆管癌标志物可能均难以提高胆管癌的早期诊断率。
小结
本课题从比较蛋白质组学的主要方法——双向凝胶电泳技术着手，以合并梗阻性黄疸的胆管癌和胆管结石患者为研究对象，筛选出两种疾病血清中的差异蛋白质，并进行质谱鉴定。根据文献研究结果，选择在胆管癌患者血清表达增强的α_1-抗胰蛋白酶和胆管结石患者血清中表达增强的PRO2619、转铁蛋白，应用免疫组织化学方法进行组织学验证，分析它们与胆管癌发生和演进的关系；然后选择α_1-抗胰蛋白酶和转铁蛋白进行血清学验证，分析它们在胆管癌诊断中的应用价值。本研究获得以下结论：
1．本课题以梗阻性黄疸患者的血清为研究对象，应用双向凝胶电泳技术分离并鉴定差异表达的血清蛋白。结果显示，通过相应的标本预处理技术，双向凝胶电泳方法可以有效的筛选出梗阻性黄疸患者血清中的差异蛋白，其中有些蛋白可能与胆管癌的发生发展有关，且有可能是胆管癌发生中的早期分子事件。
2．通过免疫组织化学技术，对筛选出的部分差异蛋白进行了组织学验证，分析α_1-抗胰蛋白酶、PRO2619和转铁蛋白在胆管癌发生发展中可能所起的作用。
3．通过免疫散射比浊法对筛选出的部分差异蛋白进行了血清学验证，并与传统血清学指标和影像学方法进行对比，分析α_1-抗胰蛋白酶和转铁蛋白在胆管癌临床诊断中的应用价值。
4．通过双向凝胶电泳-质谱技术，我们可以从胆管癌患者的血清中筛选出较多的肿瘤标志物，但目前筛选出的任何单一胆管癌标志物尚难以提高胆管癌的早期诊断率；血清蛋白质组合诊断模式和联合应用这些肿瘤标志物开发出微流控检测芯片，可能是未来提高胆管癌早期诊断率的新途径。
Foreword
The 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'
Serum
Objective 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~(TM)gelscanner 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 Protein
Objective 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.
Results
1.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.
Conclusion
1.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 Diagnoses
Methods
Objective 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.
Methods
1.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.
Results
1.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%.
Conclusion
1.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.
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