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背景:双向电泳对蛋白质混合物的分离和分析精确而有效,已成为研究蛋白质组最有价值的核心技术之一;以小鼠模型为研究对象,模拟人体疾病状态,是研究心血管疾病的重要手段之一。建立并优化小鼠心肌蛋白质组双向电泳技术将为以后的心脏疾病研究奠定基础。目的:建立并优化小鼠心肌蛋白质组双向电泳技术。设计:以小鼠为研究对象,观察对比研究。单位:南方医科大学附属南方医院病理科。材料:实验于2004-02/09在南方医科大学附属南方医院病理科完成。选取4～5周龄雄性无特殊病原体级BABL/c小鼠12只,体质量12～15g,由南方医科大学动物中心提供。方法:在麻醉状态下脱臼处死小鼠,取心脏,对不同的蛋白提取方法、上样缓冲液、范围的固相pH胶条、蛋白上样量、染色方法等进行对比分析,扫描并数字化,再进行图像分析。建立并优化实验中的各个环节。主要观察指标:蛋白分离的效果和实验的可重复性。结果:心肌特异的裂解方案能在pH4～7胶条上分离约910个蛋白点;银染上样量200μg,考染1000μg较为适宜;正常小鼠心肌组织在17cmpH3～10L的胶上可分离约(920±30)个蛋白点,pH4～7L的胶条上,可分离(880±30)个左右蛋白点;蛋白点平均匹配率为86.9%。结论:实验结果显示,对小鼠心肌组织蛋白质双向电泳各方面条件进行调整和优化,建立了相对稳定的心肌蛋白分离方法。
Abstract BACKGROUND: The separation and analysis of protein mixtures by two-dimensional electrophoresis are both accurate and effective. It has become one of the most valuable core technologies for studying proteomics. Using mouse model as a research object to simulate human disease status is an important method to study cardiovascular diseases one. Establishment and optimization of mouse myocardial proteomics two-dimensional electrophoresis technology will lay the foundation for the future of heart disease research. Objective: To establish and optimize the mouse myocardial proteome two-dimensional electrophoresis technology. Design: Mouse as the research object, observe the comparative study. Unit: Nanfang Hospital, Southern Medical University, Department of Pathology. MATERIALS: The experiment was performed in Department of Pathology, Nanfang Hospital Affiliated to Southern Medical University from February to February in 2004. Twelve male BABL / c mice with no specific pathogen at 4 ~ 5 weeks of age were selected and their body weight was 12 ~ 15g, which was provided by Animal Center of Southern Medical University. Methods: Mice were sacrificed by anesthesia, hearts were removed and different protein extraction methods, loading buffer, range of solid-phase pH strips, protein loading and staining methods were compared and analyzed. After scanning and digitizing, Perform image analysis again. Establish and optimize all aspects of the experiment. MAIN OUTCOME MEASURES: The effect of protein isolation and reproducibility of the experiment. Results: Myocardial specific lysis protocol could separate about 910 protein spots on pH 4-7 strips; 200μg silver stain was suitable for 1000μg staining; normal mouse myocardium was detached about 17cmpH3-10L (920 ± 30) spots and pH4 ~ 7L spots. The average spot-matched percentages of protein spots were 86.9%. CONCLUSION: The experimental results show that the conditions of myocardium protein two-dimensional electrophoresis in mice are adjusted and optimized, and a relatively stable myocardial protein separation method is established.