目的研究过氧化氢处理体外培养小鼠晶状体蛋白渗漏情况，并对其所渗漏的蛋白进行鉴定和生物信息分析。
方法分离42只健康成年雄性C57BL/6J小鼠双眼完整晶状体，任意合并28个晶状体作为一个样本，共3个样本。体外培养小鼠晶状体，以含2 mmol/L过氧化氢培养液进行培养，构建体外白内障模型。以空白培养液作为对照组。收集培养24 h上清液进行聚丙烯酰胺凝胶电泳分析。采用液相质谱联用对上清液蛋白质进行结构鉴定并采用无标记定量质谱法定量分析高丰度蛋白。采用METASCAPE数据库对鉴定的蛋白进行生物信息分析。采用多重微珠免疫分析系统测定上清液中细胞因子的含量。
结果过氧化氢处理24 h，所有晶状体均混浊且囊膜完整。上清液中总蛋白质量浓度为(3.73±0.59)μg/μl。聚丙烯酰胺凝胶电泳分析结果显示，蛋白条带主要位于相对分子质量35 000以下。质谱分析共鉴定出675种蛋白，其中包括16种晶状体蛋白，占总蛋白丰度的(86.1±0.8)%；蛋白涉及的生物学过程主要包括细胞-细胞黏附、细胞转化、氧化还原、抑制细胞凋亡和蛋白质转运；涉及的分子功能主要包括水解酶活性、氧化还原酶活性、催化活性、翻译起始因子活性和GTPase活性；亚细胞定位主要为细胞质、外泌体、细胞核、质膜和胞外间隙。基于抗体的多重微珠免疫分析结果显示，体外培养上清液中可定量检测到9种细胞因子，包括在眼内液高表达的单核细胞趋化蛋白-1和转化生长因子-β 2。
结论过氧化氢处理体外培养的晶状体存在蛋白质渗漏，这些渗漏蛋白可能对眼内其他组织的代谢、功能产生影响。
ObjectiveTo study the protein leakage of mouse lens treated with hydrogen peroxide in vitro, and to identify and carry out bioinformatics analysis of the leaked proteins.
MethodsIntact binocular lenses of 42 healthy male C57BL/6J mice were enucleated following sacrifice, and 28 lenses were arbitrarily combined as one sample, with 3 samples in total.Mouse lenses were cultured with medium containing 2 mmol/L hydrogen peroxide for 24 hours to establish a cataract model in vitro.Blank medium M-199 was taken as control sample.Supernatant was subjected to polyacrylamide gel electrophoresis (PAGE). The structure of the supernatant proteins was identified by liquid chromatography coupled with tandem mass spectrometry, and the high-abundant proteins were quantitatively analyzed by label-free quantitative mass spectrometry.The biological information of the identified proteins was analyzed by METASCAPE database.Content of cytokines in the supernatant was determined using the multiplex bead immunoassay system.This study protocol was approved by an Ethics Committee of The First Affiliated Hospital of Chongqing Medical University (No.2019-173), and the use and care of experimental animals complied with the ARVO statement.
ResultsAfter 24-hour culturing, the lenses became turbid, and the capsules were intact.The protein concentration in the supernatant was (3.73±0.59)μg/μl.PAGE analysis of the supernatant protein profile revealed that protein bands were below 35 000.A total of 675 proteins were identified with mass spectrometry.Sixteen crystallin proteins accounted for (86.1±0.8)% of the total protein abundance; the biological processes those proteins participated in mainly included cell-cell adhesion, cell transformation, oxidation-reduction, inhibition of apoptosis, and protein translocation; the molecular functions of those proteins mainly involved the activity of hydrolase, oxidoreductase, catalysis, translation initiation factor, and GTPase; the subcellular localization of proteins were mainly in cytoplasm, extracellular exosome, the nucleus, the plasma membrane, and the intercellular space.The results of an antibody-based multiplex bead immunoassay presented 9 cytokines in the supernatant, which included monocyte chemotactic protein-1 and transforming growth factor-β 2 highly expressed in intraocular fluid.
ConclusionsThere are protein leakages in lens treated with hydrogen peroxide in vitro, and it may have influences on metabolism and/or functions of other intraocular tissues.
李果,陈颖,严宏,等. 过氧化氢处理体外培养小鼠晶状体上清液的蛋白组学分析[J]. 中华实验眼科杂志,2021,39(05)：382-387.
DOI:10.3760/cma.j.cn115989-20200730-00546版权归中华医学会所有。
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