国人胃癌蛋白质组分析中双向凝胶电泳技术建立及优化

On the established and optimized by the two-dimensional gel electrophoresisin technique in the proteome analysis of Chinese stomach cancer

目的 初步建立、优化用于胃癌蛋白质组研究的双向凝胶电泳技术和计算机图像分析技术 ,提高其分辨率及重复性 ,得到一种分析胃癌蛋白质组双向凝胶电泳新方法。方法 提取胃癌组织中蛋白质 ,对以固相pH梯度为第一向双向电泳的关键因素与环节 ,如样品处理、电泳参数和凝胶浓度进行一系列优化。进一步采用双向凝胶电泳分离胃癌组和正常组总蛋白质 ,银染显色、图像分析软件分析电泳图谱。结果 重复性实验结果显示同组样品在 3次不同实验中所得蛋白质斑点数目相对标准差 (变异系数平均值 % ) :胃癌组和正常组分别为 2 3.0 0± 10 .11、2 0 .33± 9.90。变异系数范围 (% ) :胃癌组和正常组分别为 3.80～ 6 0 .10、2 .70～ 5 6 .89。同一蛋白质斑点在 3次实验中等电点、分子量的相对标准差分别为 8.76 %± 5 .14 % ,13.0 0 %± 4 .2 2 %和 10 .84 %± 9.16 %。获得了较好的分辨率和重复性的双向凝胶电泳图谱。结论 蛋白质组双向电泳的建立及优化表明适合于胃癌蛋白质组差异表达研究。

Objective Preliminary establish and optimize the two-dimensional gel electrophoresisin(2-DE) and computer image analysis technique for the proteome research of the stomach cancer,increase its resolution and its repetition,and gain a kind of new method to analyze stomach cancer proteome.Methods The protein of stomach cancer organization is extracted.lmmobilized pH gradient is taken as the key factor and link in the isoelectric point focus.For instance,sample handle,electrophoresis parameter and gel concentration are get on with a series of optimization.2-DE is further adopted to separate the total protein of normal term and of stomach cancer term.And then the 2-DE chart is analyzed by silver and image analysis software.Results The result of repetitive experiments shows that in three different experiments the amount of protein spots of the same term sample deviates from the relative standard as following (the average of variation coefficient %):Stomach cancer and the normal term is 23.00±10.11 and 20.33±9.90 respectively.(the range of variation coefficient %):Stomach cancer and the normal term is 3.80～60.10 and 2.70～56.89 respectively.The isoelectric point and molecular weigh of the same protein spots in three times experiments deviates from relative standard as following:8.76%±5.14%,13.00%±4.22% and 10.84%±9.16% respectively.Therefore,better resolution and repetitive 2-DE chart are obtained.Conclusion The establishment and the optimization of proteome 2-DE are qualified to the expression study of stomach cancer proteome differentials.