应用微量蛋白质定量分析评估临床样本炎性反应状态  

Quantitative analysis of trace-level proteins for assessing inflammation through clinical sampling

作　　者：叶雨昕 李慧娴 阎涛 张赟 孙玉琳 YE Yuxin;LI Huixian;YAN Tao;ZHANG Yun;SUN Yulin(State Key Laboratory of Molecular Oncology,National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,CAMS&PUMC,Beijing 100021,China;Department of Anesthesiology,National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,CAMS&PUMC,Beijing 100021,China)

机构地区：[1]国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院分子肿瘤学国家重点实验室,北京100021 [2]国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院麻醉科,北京100021

出　　处：《基础医学与临床》2024年第5期690-698,共9页Basic and Clinical Medicine

基　　金：国家自然科学基金(82073327);中国医学科学院医学与健康科技创新工程经费(2021-I2M-1-066);国家重点研发计划“中医药现代化”重点专项(2023YFC3503225)。

摘　　要：目的比较实时荧光定量PCR(qPCR)和微量蛋白质定量分析方法在乳腺癌患者外周血单核细胞中炎性相关因子caspase-1和IL-1β表达检测中的效果差异。方法收集10例乳腺癌患者的术前、术后2 h配对外周血样本各约6 mL,分离出外周血单核细胞并通过脂多糖活化;分别用qPCR和微量蛋白质定量分析方法,检测caspase-1和IL-1β在单核细胞中的mRNA和蛋白质表达水平,并使用了β-actin进行了数据归一化,比较两种检测方法结果的异同,并评估其优缺点。结果绝大部分患者经过手术应激后外周血单核细胞数量明显增加。在mRNA水平上,与术前相比,术后caspase-1和IL-1β在50%(5/10)和40%(4/10)的患者中呈现升高趋势,其余表现为降低(分别为40%和50%)或改变甚微无统计学意义(均为10%)。而微量蛋白质定量分析结果显示,与术前相比,术后caspase-1和IL-1β蛋白在60%(6/10)和60%(6/10)的患者中呈现升高趋势,其余表现为降低(分别为30%和20%)或改变较小无统计学意义(分别为10%和20%),且80%的患者在术后出现了明显增加的caspase-1和IL-1β活性切割电泳条带。重要的是,有30%和40%的患者手术前后caspase-1和IL-1βmRNA和蛋白质水平变化不一致。结论微量蛋白质定量分析方法具有样本用量少、灵敏度高、重复性好、操作相对简单等优点,更适于临床样本的炎性反应状态评估。Objective To compare the effectiveness between qPCR and trace-level proteins quantitative analysis in detecting the expression of inflammation-associated factors caspase-1 and IL-1βin peripheral blood monocytes from breast cancer patients.Methods Pre-and post-operation peripheral blood samples were collected from 10 breast cancer patients.Monocytes were subsequently isolated and activated by incubation with lipopolysaccharide.The mRNA and protein expression level of caspase-1 and IL-1βin monocytes were examined using qPCR and trace-level proteins quantitative analysis,respectively,and the data were normalized byβ-actin.The differences between the two detection methods were compared and evaluated for strengthens and weakness.Results The vast majority of patients showed a notable increase in the number of peripheral blood monocytes after surgical operation.At the mRNA level,compared to preoperative levels,postoperative caspase-1 and IL-1βwere both increased in 50%(5/10)and 40%(4/10)of patients,respectively,while the rest showed either a decrease(40%and 50%,respectively)or no significant change(both 10%).In contrast,the results of trace-level proteins quantitative analysis revealed that 60%(6/10)of patients exhibited increased level of postoperative caspase-1 and IL-1βproteins,while the remaining showed a decrease(30%and 20%,respectively)or no significant change(10%and 20%,respectively).Additionally,50%of patients displayed a significant increase in caspase-1 and IL-1βactive cleavage bands by electrophoresis postoperatively.Notably,inconsistent changes in caspase-1 and IL-1βmRNA and protein level after surgery were observed in 30%and 40%of patients respectively.Conclusions The trace-level proteins quantitative analysis is recommended due to its advantages including minimal sample amount requirement,high sensitivity,good repeatability and relatively simple operative procedure.Therefore,it is particularly suitable for assessing the inflammatory status of of patients through clinical sampling of peripheral blood

关 键 词：炎性相关因子 CASPASE-1 IL-1Β 微量蛋白质定量分析系统 实时荧光定量PCR(qPCR) 

分 类 号：R331[医药卫生—人体生理学]