人胰液蛋白质组的双向电泳方法初步研究

目的 在已有实验基础上进一步建立优化的用于人胰液差异蛋白质组研究的双向凝胶电泳(two-dimensional electrophoresis,2-DE)方法;探讨利用混合胰液进行胰腺疾病的胰液差异蛋白质组学的可行性.方法 通过ERCP术中放置鼻胰管引流收集5例胰腺癌(其中2例手术病理分别为胰腺中分化导管腺癌及导管内乳头状瘤癌变,另外3例经影像学检查结合临床诊断)和6例慢性胰腺炎胰液标本,取其中一例慢性胰腺炎胰液标本,通过改进胰液标本的处理方法、泡胀液的组成和一向等电聚焦条件后进行双向凝胶电泳,并和先前实验条件下的胰液2-DE图谱进行比较分析;同时利用优化后的双向凝胶电泳方法比较单份胰液和同病种混合胰液标本的2-DE图谱.结果 应用优化的2-DE方法,在胰液蛋白质加样量为200 μg时,可见凝胶上约有280个蛋白质点,有较好分辨率,较原条件下电泳图谱有较大改进.单份胰液和同病种混合胰液标本的2-DE图谱有较高程度的相似性(>75%).结论 优化后的胰液2-DE方法切实可行,通过各不同胰腺疾病混合胰液的2-DE图谱差异比较可为胰腺疾病的胰液差异蛋白质组学研究建立良好的基础.

Preliminary study on two-dimensional gel electrophoresis of human pancreatic juice for proteome analysis

Objective To establish an optimal method of two-dimensional gel electrophoresis (2-DE) for proteomic analysis of human pancreatic juice (pj) on the basis of former work and explore the feasibility of differential proteomic analysis of human pancreatic juice by the use of mixed pancreatic juice samples. Methods Pj samples from 5 pancreatic cancer patients (two cases were pathologically diagnosed moderately differentiated adenocarcinoma and intraduetal papilloma canceration respectively,three cases were diagnosed by inageology and clinical manifestations) and 6 chronic pancreatitis pa-tients were included in this study. Pj samples were collected by naso-pancreatic drainage during endo-scopic retrograde cholangiopancreatography. 2-DE was conducted on one pj sample of chronic panerea-titis by improving sample preparation techniques. Compositions of rehydration solutions and methods of isoelectric focusing and 2-DE maps were compared with those got from former 2-DE method. 2-DE method and 2-DE maps of single pj sample and mixed pj samples of the same pancreatic disease were also compared by the optimized method of 2-DE. Results With the improved 2-DE method and when 200 μg protein of pancreatic juice was loaded, about 280 protein spots could be successfully separated with better differentiation rate as compared with the former method. There was higher similarity (>75%)between the 2-DE maps of single pj sample and mixed pj samples of the same pancreatic disease. Conclusion Optimized method of 2-DE has reliable effect and the comparison of 2-DE maps of mixed pj samples of different pancreatic diseases would become the basis of differential proteomic analysis of human pancreatic juice.