前处理方法及冻融次数对尿液蛋白标志物质量影响的探究  

Effects of pretreatment and freeze-thaw cycles on urinary biomarkers

作　　者：李启沅 何旭珩 王县 刘小盼 孙建波 廖秋燕 尹洁芳 贾佳 唐中湘 李雪 LI Qiyuan;HE Xuheng;WANG Xian;LIU Xiaopan;SUN Jianbo;LIAO Qiuyan;YIN Jiefang;JIA Jia;TANG Zhongxiang;LI Xue(China National GeneBank,BGI-Shenzhen,Shenzhen Guangdong 518083,China)

机构地区：[1]深圳华大基因研究院国家基因库,广东深圳518083

出　　处：《转化医学杂志》2017年第5期257-260,共4页Translational Medicine Journal

基　　金：深圳市科技计划项目(JCYJ20160510141910129)

摘　　要：目的探究不同前处理方法及冻融次数对尿液样本蛋白质量的影响。方法取样后的尿液样本经过离心与不离心处理后,分别于室温及4℃冷藏放置4、12、24、48、72 h,及在-80℃反复冻融1、3、5、10次后,经过桥式酶联免疫吸附测定方法检测丛生蛋白、肾损伤分子-1、胱抑素C及视黄醇结合蛋白4种蛋白的质量浓度变化。结果经离心处理的样本,在室温或4℃冷藏放置48 h后,丛生蛋白质量浓度显著降低。未经离心处理的样本,室温放置24 h后,丛生蛋白质量浓度显著降低;4℃冷藏条件下蛋白相对稳定,在放置72h后显著降低。4种蛋白-80℃反复冻融1、3、5、10次后,质量浓度均无显著变化;未经离心处理样本,蛋白质量浓度较离心处理样本高。结论在冷冻保存前,尽量将尿液样本先放置在4℃环境下处理或暂存,且暂存时间不超过72 h;若室温放置,时间不超过24 h;且根据研究用途决定是否离心。样本反复冻融10次对尿液蛋白质量无明显影响,可以根据检测需求及实际条件,确定样本是否分装及分装管数等。Objective Aimed to explore the effects of different pretreatment methods and freeze-thaw cycles on the quality of various urinary biomarkers.Methods Centrifuged or uncentrifuged urine samples were processed and stored at room temperature of 4℃for 4,12,24,48 and 72 hours with freeze-thaw for 1,3,5,10 cycles respectively.Then four urinary biomarkers including clusterin,kidney injury molecule 1(KIM-1),cystatin C and retinol binding protein(RBP)were detected by enzyme-linked immune sorbent assay(ELISA).Results After stored at room temperature for 48 hours,the clusterin biomarker from centrifuged urine samples showed a significant reduction in concentration.While for the uncentrifuged urine samples,the cluster in biomarker showed a significant reduction in concentration after stored at room temperature for 24 hours or 4℃for 72 hours.After multiple freeze-thaw cycles,all 4 urinary biomarkers were stable,and the initial concentrations were higher for those urinary biomarkers from uncentrifugated urine samples.Conclusion Before detection or cryopreservation,urine samples were recommended to store at 4℃for maximum period of 72 hours,and stored at room temperature for no more than 24 hours.No significant degradation was detected for the four urinary biomarkers after freeze-thaw for 10 cycles,and the urine samples could be aliquoted and freezed based on research requirements.

关 键 词：尿液蛋白标志物 尿液储存 标志物质量 

分 类 号：Q592.2[生物学—生物化学]