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 HepPar1、CK19、CD34、Vimentin 蛋白表达情况，Western blot 检测组织样本冷缺血 0、6 h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 RNA RIN 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、6 h 后 HepPar1、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。
Effects of cold ischemic time on the quality of liver tissue samples in different disease types
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 cell morphology after 0 and 6 h of cold ischemia by HE staining of paraffin sections.Conclusion Cold ischemia time has a significant effect on the quality of nucleic acids in liver tissue samples,and high-quality liver tissue samples can be obtained by shortening the cold ischemia time.
万方数据
维普
