冷缺血时间对不同疾病类型肝组织质量的影响  

Effects of cold ischemic time on the quality of liver tissue samples in different disease types

作　　者：马婷婷[1] 毕森盛 曹磊 印志琪[2] 段可然 王政禄[1] 郑虹[3,4] MA Ting-ting;BI Sen-sheng;CAO Lei;YIN Zhi-qi;DUAN Ke-ran;WANG Zheng-lu;ZHENG Hong(Biobank,Tianjin First Central Hospital,Tianjin 300192,China;Pathology Department,Tianjin First Central Hospital,Tianjin 300192,China;Oriental Organ Transplant Center,Tianjin First Central Hospital,Tianjin 300192,China;Chinese Academy of Medical Sciences,Key Laboratory of Transplantation,Tianjin 300192,China)

机构地区：[1]天津市第一中心医院生物样本资源共享中心,300192 [2]天津市第一中心医院病理科,300192 [3]天津市第一中心医院器官移植研究院,300192 [4]中国医学科学院移植重点实验室,天津300192

出　　处：《中国医药生物技术》2024年第1期13-21,共9页Chinese Medicinal Biotechnology

基　　金：天津市卫生健康委员会科技项目(ZC20065);天津市自然科学基金面上项目(22JCYBJC01230)。

摘　　要：目的 高质量的肝脏组织样本对肝脏疾病的研究至关重要,冷缺血时间(CIT)是影响组织样本质量的关键因素,探讨冷缺血时间与不同疾病类型肝组织样本质量的相关性,为获得高质量肝组织样本提供标准。方法 选取天津市第一中心医院进行手术治疗的肝癌(HCC)和胆道闭锁(BA)患者各30例,手术切除肝组织后,室温放置0、0.25、0.5、1、2、3、5和6 h后,分别从基因水平、蛋白水平及形态学水平对组织质量进行检测,提取总RNA及基因组DNA,分别检测浓度、纯度及完整性(RIN/DIN值),荧光定量PCR检测组织样本冷缺血0、6 h后ALB基因表达水平;免疫组化方法检测组织样本冷缺血0、6 h后Hep Par1、CK19、CD34、Vimentin蛋白表达情况,Westernblot检测组织样本冷缺血0、6h后ALB蛋白表达水平;HE染色观察组织样本冷缺血0、6 h后组织及细胞形态学变化。结果 DNA产量、RNA纯度和RNARIN随时间变化,时间效应具有统计学意义。组间效应不显著,表明肝癌和胆道闭锁组间没有差异。DNA产量和RNA RIN的交互效应有统计学意义,说明时间的影响随着组别不同而有所不同。与冷缺血0 h相比,肝癌、胆道闭锁组织样本冷缺血6 h后ALB基因表达水平降低,差异具有统计学意义(P <0.05);免疫组化方法检测组织样本冷缺血0、6h后Hep Par1、CK19、CD34、Vimentin蛋白表达情况均无变化,Western blot检测显示组织样本冷缺血0、6 h后ALB蛋白表达水平无显著变化;石蜡切片HE染色显示样本冷缺血0、6 h后组织及细胞形态学无变化。结论 冷缺血时间对肝组织样本中核酸质量具有显著影响,缩短冷缺血时间可获得高质量肝组织样本。Objective Cold ischemia time(CIT)is one of the key factors affecting the quality of tissue samples.The study was aimed to explore the correlation between CIT and the quality of liver tissue samples in different disease types,so as to provide a standard for obtaining high-quality liver tissue samples.Methods Thirty hepatocellular carcinoma(HCC)patients and the same number of biliary atresia(BA)patients who underwent surgery in Tianjin First Central Hospital were selected,and after surgical resection of liver tissue,the tissues with HCC or BA were placed at room temperature for 0,0.25,0.5,1,2,3,5 and 6 h.The tissue quality was detected at the gene,protein and morphological levels.The total RNA and genomic DNA were extracted for the detection of concentration,purity and integrity(RNA) integrity number(RIN)/DNA integrity number(DIN).The levels of ALB mRNA and protein were detected by fluorescence quantitative PCR and Western blot after cold ischemia at 0 and 6 h.Immunohistochemistry was used to examine the protein levels of HepPar1,CK19,CD34 and Vimentin in tissue samples after 0 and 6 h of cold ischemia.Hematoxylin and eosin staining was used to detect the morphological changes of tissues and cells.Results DNA yield,RNA purity,and RIN varied over time with statistically significant time-effect.There was no significant effect between groups,indicating there were no difference between the HCC tissues and BA tissues.The differences in the interaction effect of DNA yield and RIN were statistically significant,indicating that the influence of time varied with group.The mRNA of the ALB gene in HCC and BA tissues was decreased at 6 h as compared with 0 h after cold ischemia treatment(P<0.05),but there was no significant change in the expression level of ALB protein in tissue samples after 0 and 6 h of cold ischemia.The results from immunohistochemistry showed no change in the protein expression of HepPar1,CK19,CD34 and Vimentin in tissue samples after 0 and 6 h of cold ischemia.Furthermore,there was no change in tissue and

关 键 词：冷缺血时间 肝细胞癌 胆道闭锁 肝组织质量 RNA完整性 DNA完整性 

分 类 号：R575[医药卫生—消化系统]