晚期氧化蛋白产物的制备及纯化

Preparation and purification of advanced oxidation protein products

背景:晚期氧化蛋白产物是血液透析患者免疫功能紊乱、加速性动脉粥样硬化、透析相关性淀粉样变等长期并发症的重要致病环节。但是对于晚期氧化蛋白产物的基础性研究相对较少。主要原因之一就是不能获得高纯度且具有生物学活性的晚期氧化蛋白产物。目的:制备及纯化晚期氧化蛋白产物,并对其进行鉴定,以寻求一种制备高纯度且具生物活性的晚期氧化蛋白产物的方法。设计、时间及地点:单一样本观察,于2008-09/10在解放军第三军医大学检验系临床生化教研室完成。材料:人血白蛋白为成都容生有限公司产品,Hitrap26/60sephacryl S-300为GE Healthcare产品。方法:首先纯化人血白蛋白,将纯化的人血白蛋白与次氯酸体外孵育法制备晚期氧化蛋白产物-人血白蛋白,经Hitrap26/60 sephacryl S-300分离纯化。并经变性及非变性电泳、分子筛蛋白标准鉴定其相对分子质量,经单核细胞肿瘤坏死因子α分泌实验鉴定其结构特征和生物学活性。主要观察指标:①人血白蛋白的纯化及电泳结果。②晚期氧化蛋白产物纯化及电泳结果。③晚期氧化蛋白产物-人血白蛋白促单核细胞肿瘤坏死因子α分泌的量效关系。结果:经变性及非变性电泳、分子筛蛋白标准鉴定其相对分子质量为670000,能够显著刺激单核细胞肿瘤坏死因子α的分泌,时间效应显示AOPPs-HSA(1g/L)刺激6h,单核细胞肿瘤坏死因子α的分泌量就已显著性升高,12h升至最高水平。结论:采用上述方法能够制备及纯化晚期氧化蛋白产物,且纯化后的晚期氧化蛋白产物具有生物学活性,为晚期氧化蛋白产物的进一步研究奠定了基础。

BACKGROUND： Advanced oxidation protein products （AOPPs） are a crucial pathogenic link to such long-term uremic complications in hemodialysis patients as immune system dysregulation, accelerated atherosclerosis, dialysis-related amyloidosis and so on. However, basic studies on AOPPs are relatively few, and one of the main reasons is the fact that it is difficult to obtain AOPPs with high purity and biological activity. OBJECTIVE： To prepare, purify and indentify AOPPs, with the hope of searching for a method of preparing AOPPs with high purity and biological activity. DESIGN, TIME AND SETTING： A single sample observation was completed in the Clinical Biochemistry Section of Ecsomatics Department, Third Military Medical University of Chinese PLA from September to November in 2008. MATERIALS： Human serum albumin （HSA） was provided by Chengdu Rongsheng Company Ltd. Hitrap 26/60 sephacryl S-300 was purchased from GE Healthcare. METHODS： Hypochloric acid was used in the oxidation of purified HSA to prepare in vitro the AOPPs-modified HSA （AOPPs-HSA） which was then isolated by Hitrap 26/60 sephacryl S-300. Relative molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electropheresis （SDS-PAGE）, native polyacrylamide gel electrophoresis （PAGE） and molecular weight standards. Structural features and biological activities were identified in the experiment of tumor necrosis factor α （TNF-α） release from monocytes. MAIN OUTCOME MEASURES： （1）The purification and gel electrophoresis results of HSA. （2）The purification and gel electrophoresis results of AOPPs. （3）The dose-effect relationship between AOPPs-HSA and TNF-α release from monocytes. RESULTS： The relative molecular mass of AOPPs-HSA was 670 000 according to SDS-PAGE, native PAGE and molecular weight standards. Moreover, AOPPs-HSA could encourage the release of TNF-α from monocytes. The time effects showed that TNF-α release volume significantly increased after 6 hours of stimulation by AOPPs-HSA （1 g/L） and reached a peak at hour 12. CONCLUSION： Highly purified and bioactive AOPPs can be successfully prepared by the above-mentioned method, which builds a basis for further study on AOPPs.