人胰腺癌胰液蛋白质谱差异表达初步分析

Differential proteomic analysis of pancreatic juice in pancreatic cancer patients

目的应用蛋白质组学方法分析胰腺癌胰液与慢性胰腺炎、胆总管结石患者胰液间差异蛋白质的表达，初步探索胰液中可能的胰腺癌标志物，为临床胰腺癌和慢性胰腺炎的鉴别诊断提供依据。方法内镜下逆行胰胆管造影（ERCP）放置鼻胰引流管收集患者胰液，双向凝胶电泳（2-DE）分离5例胰腺癌、6例慢性胰腺炎和3例胆总管结石患者等量？昆合胰液的蛋白质，运用图像分析软件进行比较和分析，识别差异表达蛋白质。应用基质辅助激光解析电离飞行时问质谱（matrix-assisted laser desorption／ionization time-of-flight mass spectrometry，MALDI-TOF-MS）鉴定部分差异蛋白质点。结果收集到胰液总量35～200ml，Bradford法测定蛋白质浓度为0．8～4．6μg／μI。胰腺癌、慢性胰腺炎和胆总管结石患者等量混合胰液凝胶的蛋白质点数分别为196±12、209±15和199±10，三组间两两对比后的平均匹配率均在75％以上。部分差异蛋白质点经MALDI—TOF-Ms鉴定显示：相比胆总管结石和慢性胰腺炎，转甲状腺素蛋白（TTR）二聚体支链A在胰腺癌患者胰液中表达增高；载脂蛋白A1（ApoA-1）支链A、胰石蛋白支链、Reglβ再生蛋白前体表达降低。结论人胰腺癌、慢性胰腺炎和胆总管结石患者胰液的蛋白质谱存在一定的差异，TTR、ApoA-1、胰石蛋白和Reglβ再生蛋白可能成为胰腺癌胰液中的肿瘤标志物，为临床胰腺癌和慢性胰腺炎的鉴别诊断提供依据。

Objective To analyze the differential expression of proteins among patients with pancreatic cancer, chronic pancreatitis and choledocholithiasis in order to find potential biomarkers for diagnosis of pancreatic cancer and to differentiate pancreatic cancer from chronic pancreatitis. Methods The pancreatic juice were collected from 5 pancreatic cancer patients, 6 chronic pancreatitis patients and 3 choledocholithiasis patients by nascrpancreatic drainage using endoscopic retrograde cholangiopancreatography （ERCP）. The proteins in pooled pancreatic juice were separated by two-dimensional gel electrophoresis （2-DE）. The differential expression of proteins were analyzed by image analysis software and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）. Results ① There were 35-200 ml of pancreatic juice collected, and protein concentration were ranged from 0.8 to 4.6 μg/μl. The 2-DE showed that the protein spots in pancreatic cancer, chronic pancreatitis and choledocholithiasis juice were 196 ± 12, 209 ± 15 and 199 ± 10, respectively. The matched pro- teins among three groups all exceeded 75%. ② MALDI-TOF-MS revealed that the expression of chain A of a covalent dimer of transthyretin in pancreatic cancer was up-regulated （〉2-fold） while the expressions of chain A of crystal structure of lipid-free human apolipoproteinA-1, chain of human lithostathin and regenerating islet-derived 1 beta precursor were down-regulated. Conclusions Protein spectra are different in patients with pancreatic cancer, chronic pancreatitis and choledocholithiasis. Transthyretin, apolipoproteinA-1, human lithostathin and regenerating islet-derived 1 beta might be the biomarkers of human pancreatic cancer and may be useful in distinguishing pancreatic cancer from chronic paneeatitis.