摘要
目的建立RNA干扰结肠癌SW480细胞系,并对缺氧诱导因子-1α(HIF-1α)双向凝胶电泳中的多个关键步骤进行优化,以消除横向拖尾。方法以携带针对HIF-1α两条干扰片段的pGenesil-11质粒转染SW480细胞,沉默HIF-1α。对双向凝胶电泳中样品裂解液、样品的处理方式、上样量及电泳条件等进行优化。结果转染第7天RNA干扰效果最好。RNA干扰后,SW480细胞蛋白用含有硫脲的裂解液B进行提取,以样品制备方法A进行处理后,上样200μg蛋白,延长除盐时间,聚焦50000伏小时,得到的双向凝胶电泳图基本没有横向拖尾,分离蛋白点1288±15。结论已成功建立了RNA干扰结肠癌SW480细胞系;优化的双向凝胶电泳可有效消除横向拖尾,为进一步分析、鉴定和寻找与疾病相关的蛋白质奠定了基础。
Objective To establish RNA interfering colon cancer SW480 cell line, and optimize the several key steps during two-dimensional gel electrophoresis of hypoxia-inducible factor-1α （HIF-1α） to eliminate the horizontal streak. Methods SW480 cells were transfected with recombinant plasmid pGenesil-11 carrying two specific interfering fragments to silence HIF-1α. The lytic agent of test samples, the method for treatment of samples, quantity of sample for loading as well as condition for two-dimensional gel electrophoresis were optimized. Results The effect of RNA interference 7 d after transfection was satisfactory. After the protein of SW480 cells was extracted with lytic agent B containing thiourea, treated by method A and loaded at a quantity of 200 μg for electrophoresis under the optimized condition including prolonging the time for dechlorination and focusing for 50 000 V·h, little horizontal streak was observed on electrophoretic profile, while the number of protein spots was 1 288 ± 15. Conclusion A RNA interfering colon cancer SW480 cell line was successfully established, and the horizontal streak on electrophoretic profile was effectively eliminated by optimization of condition, which laid a foundation of further analysis and identification of disease-related proteins.
出处
《中国生物制品学杂志》
CAS
CSCD
2009年第8期748-752,共5页
Chinese Journal of Biologicals
关键词
RNA干扰
结肠癌
蛋白质组学
双向凝胶电泳
横向拖尾
RNA interference
Colon cancer
Proteomics
Two-dimensional gel electrophoresis
Horizontal streak
