胰腺癌血清肿瘤标志物的蛋白质组学研究
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摘要
胰腺癌(pancreatic carcinoma, PC)是一种恶性程度高、临床表现隐匿、进展迅速、预后差的恶性肿瘤。目前尚无有效的早期诊断方法,现有的血清诊断标志物:糖链抗原19-9(CA19-9)、癌胚抗原(CEA)、糖链抗原50(CA50)等都普遍存在着敏感性差、特异性差、假阳性率高等缺陷,因此它们在胰腺癌的早期诊断过程中并不能发挥应有的作用。目前早期手术是治疗胰腺癌唯一有效的方法,但手术后患者易发生转移与局部复发,因此探索胰腺癌特异性血清肿瘤标志物以提高胰腺癌早期诊断率和预测胰腺癌复发转移显然是必要的。本研究通过蛋白质组学(proteomics)的相关技术:双向荧光差异凝胶电泳(Fluorescence 2-D Difference Gel Electrophoresis, 2D-DIGE)和基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry, MALDI-TOF-MS)技术比较分析了胰腺癌患者(分术前、术后两组)、慢性胰腺炎(chronic pancreatitis,CP)患者及健康对照组外周血清中蛋白的表达情况,筛选出了胰腺癌差异表达蛋白。
目的
应用蛋白组学相关技术分离鉴定胰腺癌差异表达蛋白,为胰腺癌血清肿瘤标志物的筛选提供参考。同时鉴定与胰腺癌复发转移相关的差异性蛋白,为临床提高对胰腺癌复发转移的早期诊断和预测胰腺癌预后提供理论实验依据。
方法
(1)2D-DIGE分离胰腺癌患者(分术前、术后组)、慢性胰腺炎患者及正常对照组外周血清中的蛋白质,图像分析识别差异表达的蛋白质点。
(2)MALDI-TOF-MS鉴定差异表达的蛋白质点,筛选出胰腺癌差异表达蛋白和与胰腺癌复发转移相关的差异表达蛋白。
结果
(1)2D-DIGE联合MALDI-TOF-MS技术共分离鉴定出7个差异表达蛋白:补体3(complement component,C3)、高度相似血清蛋白cDNA FLJ50830、结合珠蛋白(haptoglobin,HP)、载脂蛋白E(apolipoprotein E,ApoE)、补体4B1(C4B1,Homo sapiens)、TNIP2 (Isoform 1 of TNFAIP3-interacting protein 2)蛋白和血清淀粉样蛋白P成分Chain A。
(2)其中C3和cDNA FLJ50830蛋白在正常对照组中低表达,在胰腺癌术前组和术后组高表达;在胰腺癌术前组和慢性胰腺炎组的比较中,C3的表达胰腺癌术前组高于慢性胰腺炎组,cDNA FLJ50830蛋白的表达慢性胰腺炎组高于胰腺癌术前组。
(3)cDNA FLJ50830蛋白、HP、apolipoprotein E、C4B1、TNIP2和血清淀粉样蛋白P成分Chain A在胰腺癌术前组高表达,在胰腺癌术后组低表达。
结论
(1)炎症应答调节因子补体3的发现提示了炎症反应与胰腺癌的发生发展密切相关,为阐明胰腺癌的发生机制提供了新的思路。
(2)cDNA FLJ50830为新发现胰腺癌差异表达蛋白,可能成为临床胰腺癌与慢性胰腺炎鉴别诊断的新的血清学标志物。
(3)HP、apolipoprotein E、C4B1、TNIP2和血清淀粉样蛋白P成分Chain A可能成为诊断胰腺癌复发转移的血清标志物。
Pancreatic carcinoma (PC) is one of the malignant tumor, its clinical manifestations was not obvious, developed rapidly, and its prognosis is very bad. At present, there is no effective screen and early diagnostic methods. Current serum tumor biomarkers for pancreatic carcinoma including carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA) and carbohydrate antigen 50 (CA50) all have many defects such as bad-sensitivity, bad-specific, high rate of false-positive, they could not play their due role in the early diagnosis of pancreatic carcinoma. Surgical management is only effective method to treat pancreatic carcinoma, but metastasis and local recurrence were more easily appeared in patients of pancreatic carcinoma after surgery. It is necessary to identify the serum markers for pancreatic carcinoma in order to improve early diagnosis rate and predict recurrence and metastasis. In this study, fluorescence 2-D Difference gel electrophoresis (2D-DIGE) and surface enhanced laser desorption / ionization time-of-flight mass spectrometry(SELDI-TOF-MS) were be used to analyze the expression of serum protein in the pancreatic carcinoma patients ( preoperative group and postoperative group), chronic pancreatitis (CP) patients and healthy control group. Differentially expressed proteins of pancreatic carcinoma were be identified.
Objective
To identify differentially expressed proteins of pancreatic carcinoma by comparative proteomics, which provide reference to the screenings of serum tumor markers for pancreatic carcinoma. At the same time differential proteins which were associated with recurrence and metastasis of pancreatic carcinoma were also be identified, it would provide a theoretical foundation for improving early diagnosis of recurrence and metastasis of pancreatic carcinoma as well as forecasting prognosis of pancreatic carcinoma.
Methods
(1)proteins were separated from peripheral blood in pancreatic carcinoma patients (preoperative group and postoperative group), chronic pancreatitis patients and normal control group by 2D-DIGE, differentially expressed proteins spot were analyzed and described.
(2)Several proteins were be identified from the differentially expressed protein spots by SELDI-TOF-MS. It was confirmed that there were closely relationships between those proteins and pancreatic carcinoma, including the occurrence, recurrence and metastasis of pancreatic carcinoma.
Results
(1)Seven differentially expressed proteins: complement component 3(C3), cDNA FLJ50830( highly similar to Serum albumin), haptoglobin protein (HP protein), apolipoprotein E (ApoE), C4B1 (Homo sapiens), Isoform 1 of TNFAIP3-interacting protein 2(TNIP2)and Chain A (The Structure Of Pentameric Human Serum Amyloid P Component) were identified by 2D-DIGE and SELDI-TOF-MS.
(2)C3 and cDNA FLJ50830 were high expression in pancreatic carcinoma group(including preoperative group and postoperative group) compared with normal control group. The expression of C3 was higher in the preoperative group of pancreatic carcinoma than in the chronic pancreatitis group, the expression of cDNA FLJ50830 was higher in chronic pancreatitis group than in the preoperative group of pancreatic carcinoma.
(3) cDNA FLJ50830, HP protein, ApoE、C4B1、Isoform 1 of TNIP2 and Chain A (the structure of pentameric human serum amyloid P component) were higher expression in the preoperative group compared with the postoperative group of pancreatic carcinoma.
Conclusions
(1)Inflammatory response regulatory factor C3 was high expressed in pancreatic carcinoma compared with normal control group revealed that inflammatory response was closely related to the occurrence and development of pancreatic carcinoma, which would provide a new way to understand the pathogenesis of pancreatic carcinoma.
(2)cDNA FLJ50830 was a newly discovered protein differentially expressed in pancreatic carcinoma, which may become a new serum marker for the differential diagnosis of pancreatic carcinoma and chronic pancreatitis.
(3)HP, apolipoprotein E, C4B1, Isoform 1 of TNIP2 and Chain A (the structure of pentameric human serum amyloid P component ) may be the serum tumor markers which could diagnose the recurrence and metastasis of pancreatic carcinoma.
目的
应用蛋白组学相关技术分离鉴定胰腺癌差异表达蛋白,为胰腺癌血清肿瘤标志物的筛选提供参考。同时鉴定与胰腺癌复发转移相关的差异性蛋白,为临床提高对胰腺癌复发转移的早期诊断和预测胰腺癌预后提供理论实验依据。
方法
(1)2D-DIGE分离胰腺癌患者(分术前、术后组)、慢性胰腺炎患者及正常对照组外周血清中的蛋白质,图像分析识别差异表达的蛋白质点。
(2)MALDI-TOF-MS鉴定差异表达的蛋白质点,筛选出胰腺癌差异表达蛋白和与胰腺癌复发转移相关的差异表达蛋白。
结果
(1)2D-DIGE联合MALDI-TOF-MS技术共分离鉴定出7个差异表达蛋白:补体3(complement component,C3)、高度相似血清蛋白cDNA FLJ50830、结合珠蛋白(haptoglobin,HP)、载脂蛋白E(apolipoprotein E,ApoE)、补体4B1(C4B1,Homo sapiens)、TNIP2 (Isoform 1 of TNFAIP3-interacting protein 2)蛋白和血清淀粉样蛋白P成分Chain A。
(2)其中C3和cDNA FLJ50830蛋白在正常对照组中低表达,在胰腺癌术前组和术后组高表达;在胰腺癌术前组和慢性胰腺炎组的比较中,C3的表达胰腺癌术前组高于慢性胰腺炎组,cDNA FLJ50830蛋白的表达慢性胰腺炎组高于胰腺癌术前组。
(3)cDNA FLJ50830蛋白、HP、apolipoprotein E、C4B1、TNIP2和血清淀粉样蛋白P成分Chain A在胰腺癌术前组高表达,在胰腺癌术后组低表达。
结论
(1)炎症应答调节因子补体3的发现提示了炎症反应与胰腺癌的发生发展密切相关,为阐明胰腺癌的发生机制提供了新的思路。
(2)cDNA FLJ50830为新发现胰腺癌差异表达蛋白,可能成为临床胰腺癌与慢性胰腺炎鉴别诊断的新的血清学标志物。
(3)HP、apolipoprotein E、C4B1、TNIP2和血清淀粉样蛋白P成分Chain A可能成为诊断胰腺癌复发转移的血清标志物。
Pancreatic carcinoma (PC) is one of the malignant tumor, its clinical manifestations was not obvious, developed rapidly, and its prognosis is very bad. At present, there is no effective screen and early diagnostic methods. Current serum tumor biomarkers for pancreatic carcinoma including carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA) and carbohydrate antigen 50 (CA50) all have many defects such as bad-sensitivity, bad-specific, high rate of false-positive, they could not play their due role in the early diagnosis of pancreatic carcinoma. Surgical management is only effective method to treat pancreatic carcinoma, but metastasis and local recurrence were more easily appeared in patients of pancreatic carcinoma after surgery. It is necessary to identify the serum markers for pancreatic carcinoma in order to improve early diagnosis rate and predict recurrence and metastasis. In this study, fluorescence 2-D Difference gel electrophoresis (2D-DIGE) and surface enhanced laser desorption / ionization time-of-flight mass spectrometry(SELDI-TOF-MS) were be used to analyze the expression of serum protein in the pancreatic carcinoma patients ( preoperative group and postoperative group), chronic pancreatitis (CP) patients and healthy control group. Differentially expressed proteins of pancreatic carcinoma were be identified.
Objective
To identify differentially expressed proteins of pancreatic carcinoma by comparative proteomics, which provide reference to the screenings of serum tumor markers for pancreatic carcinoma. At the same time differential proteins which were associated with recurrence and metastasis of pancreatic carcinoma were also be identified, it would provide a theoretical foundation for improving early diagnosis of recurrence and metastasis of pancreatic carcinoma as well as forecasting prognosis of pancreatic carcinoma.
Methods
(1)proteins were separated from peripheral blood in pancreatic carcinoma patients (preoperative group and postoperative group), chronic pancreatitis patients and normal control group by 2D-DIGE, differentially expressed proteins spot were analyzed and described.
(2)Several proteins were be identified from the differentially expressed protein spots by SELDI-TOF-MS. It was confirmed that there were closely relationships between those proteins and pancreatic carcinoma, including the occurrence, recurrence and metastasis of pancreatic carcinoma.
Results
(1)Seven differentially expressed proteins: complement component 3(C3), cDNA FLJ50830( highly similar to Serum albumin), haptoglobin protein (HP protein), apolipoprotein E (ApoE), C4B1 (Homo sapiens), Isoform 1 of TNFAIP3-interacting protein 2(TNIP2)and Chain A (The Structure Of Pentameric Human Serum Amyloid P Component) were identified by 2D-DIGE and SELDI-TOF-MS.
(2)C3 and cDNA FLJ50830 were high expression in pancreatic carcinoma group(including preoperative group and postoperative group) compared with normal control group. The expression of C3 was higher in the preoperative group of pancreatic carcinoma than in the chronic pancreatitis group, the expression of cDNA FLJ50830 was higher in chronic pancreatitis group than in the preoperative group of pancreatic carcinoma.
(3) cDNA FLJ50830, HP protein, ApoE、C4B1、Isoform 1 of TNIP2 and Chain A (the structure of pentameric human serum amyloid P component) were higher expression in the preoperative group compared with the postoperative group of pancreatic carcinoma.
Conclusions
(1)Inflammatory response regulatory factor C3 was high expressed in pancreatic carcinoma compared with normal control group revealed that inflammatory response was closely related to the occurrence and development of pancreatic carcinoma, which would provide a new way to understand the pathogenesis of pancreatic carcinoma.
(2)cDNA FLJ50830 was a newly discovered protein differentially expressed in pancreatic carcinoma, which may become a new serum marker for the differential diagnosis of pancreatic carcinoma and chronic pancreatitis.
(3)HP, apolipoprotein E, C4B1, Isoform 1 of TNIP2 and Chain A (the structure of pentameric human serum amyloid P component ) may be the serum tumor markers which could diagnose the recurrence and metastasis of pancreatic carcinoma.
引文
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2.Real FX. A“catastrophic hypothesis”for pancreas cancer progression[J]. Gastroenterology, 2003;124(7):1958-1964.
3. Koprowski H, Steplewski Z, Mitchell K ,et al. Colorectal carcinoma antigens detected by hybridoma antibodies[J]. Somatic Cell Genet 1979; 5(6): 957-971.
4. Levy C, Lymp J, Angulo P, et al. The value of serum CA 19-9 in predicting cholangiocarcinomas in patients with primary sclerosing cholangitis[J]. Dig Dis Sci 2005;50(9):1734-1740.
5. Gold P, Freedman SO. Specific carcinoembryonic antigens of the human digestive system[ J ]. J Exp Med, 1965;122 (3) :467-481.
6.沈霞,李稻.临床免疫学与免疫学检验[M ].北京:人民军医出版社, 2002: 196
7. Baeckstrom D, Hansson GC, Nilsson O, et al. Purification and char2 acterization of amembrane2bound and a secreted mucin2type glycopro2 tein carrying the carcinoma2associated sialyl2Lea ep itope on distinct core proteins[J]. J Biol Chem, 1991;266 (32) :21537-21547.
8. Zhang LZ, Gong YF, Tu ZX.Quantitative determination of serum levels of CA242 in patients with pancreatic cancer and its significance[J]. Chin J Pancreatol 2002;2(10):20-21
9. Ventrucci M ,Cipolla A , Racchini C, et a1.Tumor M 2-pyruvate kinase, a new metabolic marker for pancreatic cancer[J].Dig Dis Sci.2004;49(7-8):1149-1155.
10. Zhou G, Li H, DeCamps D, et al. 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers[J]. Mol Cell Proteomics, 2002; 1(2):117-124.
11. Liotta LA , FerrariM , Petricoin E. Clinical proteomics: written in blood [J]. Nature,2003;425(6961):905.
12.Gharbi S,Gaffney P,YangA, et a.l Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system[J]. Mol Cell Proteomics,2002;1(2):91-98.
13.Mao YS, Zhang YS. Discovering tumor proteomic patterns in biological fluids using SELDI-TOF strategy [J].Oncology prog, 2004;2(3):203-209.
14.Petricoin EF, Ardekani AM, Hill BA et al. Use of proteomic patterns in serumto identifyovarian cancer[J]. Lancet, 2002;359(9306):572-577.
15.Li J, Zhang Z, Rosenzweig J, et al. Proteomics and bioinformatics approaches for identification of serum biomarkers to detect breast cancer [J]. Clin Chem, 2002;48(8):1296-1304.
16.Zhang GQ, Du J, Pang D. Detection and clinical significance of serum proteomic patternsof breast cancers by surface enhanced laser desorptionionization time of flight mass spectrometry[J].Clin J Oncol, 2006;28(3):204-207.
17.Adam BL, Qu Y, Davis JW, et al. Serum protein fingerprinting coupled with a pattern matching algorithm distinguishes prostate cancer frombenign prostate hyperplasia and healthy men [J]. Cancer Res,2002;62(13):3609-3614.
18.Hugli TE. Structure and function of the anaphylatoxin [J].Springer Semin Immunopathol, 1984; 7: 193-219.
19.ScholzW, McclurgMR, Cardenas GJ, et al. C5a-mediated release of interleukin26 by human monocytes[ J ]. Clin Immunol Immunopathol, 1990; 57(2): 297-307.
20. Szalai AJ, van Ginkel FW, Wang Y, et al. Complement-dependent acute-phase expression of C-reactive protein and serum amyloid P-component[J]. J Immunol. 2000;165(2):1030-1035.
21. Wang W, Abbruzzese JL, Evans DB, et al. The nuclear factor-kappa B Rel A transcription factor is constitutively activated in human pancreaticadenocarcinoma cells[J]. Clin Cancer Res. 1999;5(1)):119-127.
22. Habermann JK, Roblick UJ, Luke BT, et al. Increased serum levels of comp lement C3a anaphylatoxin indicate the p resence of colorectal tumors[ J ]. Gastroenterology, 2006; 131 (4):1020-1029.
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