中文摘要：

目的：建立和优化重症肌无力（MG）胸腺组织蛋白质组双向电泳技术方法。方法：提取MG患者（n=3）增生型胸腺组织蛋白，以固相pH梯度胶条做第一向等电聚焦电泳，SDS聚丙烯酰胺凝胶垂直电泳为第二向。在以上过程中分别对蛋白提取、等电聚焦程序（A、B、C、D）和凝胶染色方法等各个实验环节进行控制和优化，利用PDQuest7．1．1软件分析获得的二维凝胶图像。结果：①采用一步法提取胸腺组织蛋白质，第一向等电聚焦采用7cm IPG胶条，聚焦程序选择B，硝酸银染色。3例标本重复4次，共获12幅二维凝胶图像。随机选取1例标本的2幅图像，利用软件对2幅中的蛋白点进行匹配，匹配率为82．1％；3份样品共12幅凝胶图像的对比，获得3个共同存在的标志蛋白点，相对分子质量和等电点分别为：A点（82700，7．0）、B点（50700，5．76）和C点（33 300,502）。②进一步优化上述实验条件，选择1例标本，采用两步法提取胸腺组织蛋白质，选用17cm IPG胶条，聚焦采用程序D，考马斯亮蓝染色，获得的二维凝胶图像与一步法（其他条件相同）比较。一步法可检测到蛋白质点326个，两步法可检测到562个蛋白点。结论：①建立了MG胸腺组织蛋白质组提取的方法。采用两步法可以更有效地提取胸腺组织蛋白质组。②利用优化后的实验方法，获得了MG胸腺组织蛋白二维凝胶图像，为开展MG胸腺组织蛋白质组学研究打下了基础。

英文摘要：

Aim ： To establish and optimize the technique of two-dimensional gel electrophoresis （2-DE） for proteomic analysis of thymus tissue in patients with myasthenia gravis （MG）. Methods： The proteins from thymus tissue of 3 cases of MG were extracted. The 2-DE system with the first dimension using isoelectrofocusing electrophoresis and the second dimension using SDS-poly-acrylamide gel electrophoresis was empl,oyed to obtain the 2-DE gel maps. The conditions of the above procedures were optimized. ReSUltS： To extract the proteins by one-step method, employ the 2-DE system with the first dimension using 7 cm IPG ready strips, isoelectrofocusing procedure B, AgNO3 staining. The experiments were perfonmed with 3 samples repeated 4 times. A total of 12 2-DE gel maps were obtained. Two gel maps from 1 sample were chosen. The matching rate of protein spot was 82.1%. By comparing the 12 gel maps, we obtained 3 marker protein spots in common, and their relative molecular weight and isoelectric point were： A （82 700,7.0） , B （50 700, 5.76）, C （33 300, 5.02）. To further optimize the experimental conditions, we chose a sample and extracted the proteins by twostep method, using 2-DE system with 17 cm IPG ready strips, coomassie brilliant blue R-250 staining. The obtained 2-DE gel maps were compared with one-step method （the other experimental conditions were the same）. The results showed that the obtained protein spot was 326 by one-step method and 562 by two-step method. Conclusion ： The method of extracting thymus tissue protein was established. Instead of extracting thymus proteins using lysate Ⅰ, the protein spots in 2-DE gel map were significantly increased using lysate Ⅰ and Ⅱ; by using the optimized method described above, satisfactory 2-DE maps of MG hyperplastic thymus tissue has been obtained, which lays a foundation for the further study of the proteome of MG hyperplastic thymus tissue.