不同时间下超低温保存肝癌组织细胞RNA完整性的差异分析  

Analysis of RNA integrity of hepatocellular carcinoma cells with different cryopreservation times

作　　者：曹国强[1] 张峰[1] 陈雷鸣[1] 陈海[1] 李彪 冯晓文[1] 李红春[2] 谢海洋[1] 周琳[1] Cao Guoqiang;Zhang Feng;Chen Leiming;Chen Hai;Li Biao;Feng Xiaowen;Li Hongchun;Xie Haiyang;Zhou Lin(First Affiliated Hospital,Key Laboratory of Combined Multiorgan Transplantation,Ministry of Public Health,Key Laboratory of Organ Transplantation,Zhejiang University School of Medicine,Hangzhou 310003,China;Shenzhen Third People’s Hospital,Division of Hepatobiliary Surgery,Shenzhen 518112,China)

机构地区：[1]浙江大学医学院附属第一医院卫生部多器官联合移植研究重点实验室浙江省器官移植重点实验室,杭州310003 [2]深圳市第三人民医院肝胆外科,518112

出　　处：《中华细胞与干细胞杂志（电子版）》2017年第1期49-53,共5页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：浙江省"十二"五基层卫生适宜技术成果转化工程重大项目(2013T301-15);深圳市三名工程科技计划项目(ZDSYS201504301534057)

摘　　要：目的研究肝癌组织细胞样本于-80℃超低温保存条件下的质量控制,探讨肝癌组织细胞样本在超低温保存中的时效性。方法随机抽取超低温新鲜冻存(24 h内)、保存3个月、6个月和1年不同时长的肝癌和对应的癌旁组织细胞各12对,HE染色评估标本取材的准确性,采用Promega自动提取核酸仪提取肝脏细胞RNA,利用Agilent 2100生物分析仪进行RIN值及28S/18S比值分析。对新鲜冻存、保存3个月、6个月以及1年的组织细胞样本的RIN值和28S/18S比值进行组间非配对t检验。结果 HE染色镜下观察发现切片组织细胞中肿瘤细胞比例达到80﹪以上,为生物样本库的合格样本。生物分析仪检测结果显示超低温新鲜冻存、保存3个月、6个月和1年的癌和癌旁组织细胞的RIN均值均>7.0,平均28S/18S比值均>1.5,组间非配对t检验显示,保存1年的肝癌组织细胞相比于新鲜冻存(7.442±0.674 vs 8.617±0.769,P=0.001)、保存3个月(7.442±0.674 vs 8.275±0.617,P=0.005)和6个月(7.442±0.674 vs 8.175±0.970,P=0.043)的组织细胞RIN值显著下降,提示存在一定程度的RNA降解,而其余各组之间差异均无统计学意义(P均>0.05)。结论在超低温条件下新鲜冻存、保存3个月、6个月的肝癌及癌旁组织细胞,其质量均可以获得有效保证,而保存1年的肝癌组织细胞的RNA虽然存在一定程度的降解,但能够满足后续临床科研的要求。Objective To evaluate the quality control system for cryopreservation of hepatocellular carcinoma cells under-80 C as time relapses.Methods Hepatocellular carcinoma tissue was frozen within 24 hours after harvesting and stored for three months,six months and one year under ultra-low temperature respectively(n=12 for each group).Hepatocellular carcinoma and paracancerous tissue were identified by HE Staining.Promega automatic nucleic acid extraction instrument was used to extract RNA from HCC cells.RIN(RNA integrity number)value and 28S/18S ratio was evaluated by Agilent 2100 Bioanalyzer.Results The percentage of tumor cells were found in more than 80%of the samples by HE staining,and were considered as qualified samples.The average RIN values and 28S/18S ratios for HCC tissue stored for three months,six months and one year were>7.0 and>1.5 respectively.Unpaired t test showed that RIN value of cells stored for one year was decreased significantly compared with that freshly collected samples(7.442±0.674 vs 8.617±0.769,P=0.001)or samples stored for three months(7.442±0.674 vs 8.275±0.617,P=0.005)and six months(7.442±0.674 vs 8.175±0.970,P=0.043),suggesting the presence of RNA degradation.There was no significant difference between the other groups(P>0.5).Conclusion Morphology for tumor cells,RIN value and 28S/18S ratio can be used as indicators for monitoring the quality of liver cancer samples.Sample quality remained good when stored at ultra-low temperatures for three months or six months,although there was a certain degree of RNA degradation for samples that stored for one year.

关 键 词：肝肿瘤 组织细胞 RNA 低温保存 

分 类 号：R735.7[医药卫生—肿瘤]