青果作用后大鼠肝星状细胞蛋白质组双向电泳技术的建立

[目的]建立青果作用后大鼠肝星状细胞蛋白质组双向电泳分离体系,提高其分辨率和重复性。[方法]青果作用于大鼠肝星状细胞,提取全蛋白,按标准条件对蛋白质进行双向电泳分离,并对各关键因素进行优化。[结果]最终选择的裂解液配方是1%TBP、4%CHAPS、0.2%Bio-Lyte、40 mmol/L Tris、8 mol/L尿素、2 mol/L硫脲;使用pH 4～7的IPG胶条进行被动水化上样,等电聚焦采用缓慢升压模式,电泳参数根据Bio-Rad公司的预设方案进行调整;使用改良硝酸银法进行蛋白质斑点染色,从而获得了满意的蛋白质组双向电泳图谱。[结论]成功建立了青果作用后大鼠肝星状细胞蛋白质组双向电泳分离体系,具有较高的分辨率和重复性,为进一步较为系统、全面、准确的评价青果及其复方制剂的作用机制和药效提供方法学基础。

Establishment of Two-dimensional Electrophoresis System for Proteomes from Rbl Treated Hepatic Stellate Cell of Rats

[ Objective ] The study aimed to establish a system of two-dimensional gel electrophoresis （2-DE） for separation of proteomes from rat HSC-T6 cells, improve its resolution and reproductivity. [Method] After the cultured HSC-T6 cells were treated by Chinese white olive, the proteins were extracted, and separated by 2-DE in standard conditions with an optimization for each important experimental parameter. [ Result ] The lysis buffer containing 1% TBP, 4% CHAPS, 0.2% Bio-Lyte, 40 mmol/L riffs, 8 mol/L urea, 2 mol/L thiourea was applied to extract proteins from cells. The passive rehydration scheme of samples was used to help the separation of large molecular weight proteins in the IPG gel. Electrophoresis parameters recommended by Bio-Rad Corporation were adjusted adequately. In the process of silver staining, a suffi- cient washing operation ensured a week background of 2-DE maps. [ Conclusion ] Under above optimized conditions, 2-DE maps with high res- olution were reproducibly obtained. Therefore a 2-DE separation system for proteomes from HSC-T6 cells was successfully established, which provide methodological basis for comprehensively, accurately evaluating the function of Chinese white olive and its compound preparation.