【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。更多还原

【Abstract】 Objective To evaluate the quality control system for cryopreservation of hepatocellular carcinoma cells under-80 ℃ 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/18 S 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/18 S 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/18 S 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.更多还原