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目的:采用蛋白质组学研究策略,研究R/S-普萘洛尔(Propranolol,PRO)的手性吸收动力学特征,建立其差异蛋白质图谱。方法:体外培养Caco-2细胞并使之在培养瓶底部融合达80%以上;将含160μmol/L的R-PRO和S-PRO的细胞培养液分别加入到培养瓶中,在培养箱中静置培养4 h;吸弃药液,用刮除法收集培养瓶中的Caco-2细胞,细胞全蛋白提取液裂解并收集Caco-2细胞全蛋白,Bradford法蛋白定量;取100μg全蛋白进行双向电泳,建立R-PRO和S-PRO的差异蛋白质图谱;对差异蛋白进行质谱鉴定和生物信息学检索,根据蛋白功能探讨R/S-普萘洛尔的手性吸收动力学特征。结果:R-PRO和S-PRO的蛋白质图谱差异不明显,对其中的明显差异蛋白质点进行质谱检测,成功鉴定到8个蛋白,分别为:GRP78、GRP75、HSP7C、HSP71、Cytokeratin 8、PIP5K、Ran、Prohibition,经生物物信息学检索,这些蛋白的功能主要涉及细胞的应急反应、能量代谢、生长繁殖等,与药物的主动吸收无直接相关。结论:成功建立了R-PRO和S-PRO的差异蛋白质图谱;R-PRO和S-PRO在蛋白质组学方面虽然存在差异,但这些蛋白与药物的主动吸收无直接相关,推测两药在体外的吸收转运无手性差异,与其它方法的研究结果一致。此外,研究中发现的差异蛋白提示,R-PRO和S-PRO在对细胞的新陈代谢方面有差异,可能与两者在体内的系统清除率的差异相关,这有待进一步研究证实。
OBJECTIVE: To study the chiral absorption kinetics of R / S-Propranolol (PRO) using proteomics strategy and to establish the differential protein profiles. Methods: Caco-2 cells were cultured in vitro and fused to more than 80% at the bottom of culture flask. The cell culture medium containing 160 μmol / L R-PRO and S-PRO was added into culture flasks respectively. Caco-2 cells were harvested and the Caco-2 cells were harvested. Total protein was extracted from the whole Caco-2 cells and quantified by Bradford method. 100 μg of whole protein was used for two-dimensional electrophoresis To establish the differential protein profiles of R-PRO and S-PRO. The differential proteins were identified by mass spectrometry and bioinformatics search, and the chiral absorption kinetics of R / S-propranolol was discussed based on the protein function. RESULTS: There were no significant differences in protein profiles between R-PRO and S-PRO. Eight of the proteins were successfully identified by mass spectrometry, including GRP78, GRP75, HSP7C, HSP71, Cytokeratin 8, PIP5K, Ran and Prohibition, which were searched by bioinformatics methods. The functions of these proteins are mainly related to the cellular emergency response, energy metabolism, growth and reproduction, and are not directly related to the active absorption of drugs. Conclusion: The differential protein profiles of R-PRO and S-PRO have been established successfully. Although there are differences in proteomics between R-PRO and S-PRO, these proteins are not directly related to the active absorption of drugs. The absorption and transport without chiral differences, consistent with the findings of other methods. In addition, the differential proteins found in the study suggest that R-PRO and S-PRO have differences in cell metabolism, which may be related to the difference in the systemic clearance between the two, which needs further study to confirm.