深低温冻存胃癌样本的核酸与蛋白质质量控制研究  

Quality control of nucleic acids and protein of freeze-preserving gastric cancer samples

作　　者：张佳年[1,2] 计骏[2] 刘炳亚[2] 朱正纲[2] 傅国辉[1] 于颖彦[2] 

机构地区：[1]上海交通大学基础医学院病理教研室,上海市200025 [2]上海交通大学医学院附属瑞金医院消化外科研究所,上海市胃肿瘤重点实验室

出　　处：《中国肿瘤临床》2016年第1期27-34,共8页Chinese Journal of Clinical Oncology

基　　金：国家高技术研究发展计划“863”项目(编号:2012AA02A504,2012AA02A203);国家自然科学基金项目(编号:81172329,81372644);上海交通大学医学院转化医学创新基金(编号:15ZH1002,15ZH4001);“985”生物样本库建设基金资助~~

摘　　要：目的：探索上海市瑞金医院低温冻存0～10年胃癌样本的核酸与蛋白质质量。方法：随机抽取2003年至2014年间库存手术切除胃癌标本24对（共48份）。1％琼脂糖凝胶电泳检测DNA与RNA纯度及完整性，同时增加了RIN值评估法检测RNA完整性；BCA法检测样本蛋白质浓度，考马斯亮蓝法测定蛋白质分子完整性。结果：将组织标本按冻存年限分成四组（〈2年、3～5年、6～8年和〉9年），各组之间DNA完整性差异无统计学意义（P〉0．05），正常胃组织的DNA降解程度比胃癌组织高（P=0．023）；四组之间的RIN值检测显示，冻存6年以上组的RIN值有明显下降（P=0．018）；各组间的蛋白质浓度无显著性差异，考马斯亮蓝法测定蛋白质分子条带完整性存在显著性差异。冻存〉9年组仅出现少量低分子量条带（平均36．5kD），冻存6～8年组尚有中等分子量（平均65．63kD）条带，冻存3～5年组有高分子量（平均127．5kD）条带，冻存短于2年组仍可见大分子量（平均160kD）条带。结论：长期低温冻存对DNA影响最小，超过5年组RNA和大分子量蛋白质降解较多，但中小分子量蛋白质保存完好。Objective： To explore the quality of inventory samples of a biobank stored in a deep freezer from 0 to over 10 years in Shanghai Ruijin Hospital. Methods： We extracted 24 pairs of stocked gastric cancer samples between 2003 and 2014. We used 1% agarose gel electrophoresis to analyze DNA and RNA purity and integrity while adding the RNA integrity number （RIN） for precise analysis. Bicinchonininc acid （BCA） assay was used for protein concentration evaluation. Coomassie brilliant blue method was used for protein integrity assay. Results： The samples were divided into four groups according to cryopreservation period （〈2 years, 3-5 years, 6-8 years, and 〉9 years）. No significant difference in DNA integrity was found between the groups （P〉0.05）; however, DNA degradation in normal gastric mucosa was faster than that in gastric cancer tissue （P=0.023）. The RIN significantly declined when the storage period was 6 years or longer （P=0.018）. No significant difference in protein concentration was observed between different groups. Using Coo- massie brilliant blue method, we found significant differences in preserved proteins with different molecular weights. Proteins with varying molecular weights were detected in the groups with the following cryopreservation periods： 〉9 years, a small number of low- molecular-weight （average 36.5 KD） proteins; 6-8 years, medium-molecular-weight （average 65.63KD） proteins; 3-5 years, high-molecular-weight （average 127.5 KD） proteins; 〈2 years, high-molecular-weight （average 160 KD） proteins. Conclusion; Cryopreservation does not exert an obvious effect on DNA. If the cryopreservation period is more than 5 years, serious degradation of RNA should occur; likewise, degradation of proteins with higher molecular weight should occur.

关 键 词：生物样本 胃癌 低温保存 核酸 蛋白质 质量控制 

分 类 号：R735.2[医药卫生—肿瘤]