Spruessel Annika, Steimann Garnet, Jung Mira, Lee Sung A, Carr Theresa, Fentz Anne-Kristin, Spangenberg Joerg, Zornig Carsten, Juhl Hartmut H, David Kerstin A
Center for Cancer Research, Israelitic Hospital, Hamburg, Germany.
Biotechniques. 2004 Jun;36(6):1030-7. doi: 10.2144/04366RR04.
The aim of this study was to determine the impact of ischemia on gene and protein expression profiles of healthy and malignant colon tissue and, thus, on screening studies for identification of molecular targets and diagnostic molecular patterns. Healthy and malignant colon tissue were snap-frozen at various time points (3-30 min) after colon resection. Gene and protein expression were determined by microarray (HG-U133A chips) and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) technology (CM10 chips, SAX2 chips, and IMAC3Ni chips), respectively. Real-time reverse transcription PCR (RT-PCR) was used for comparative measurement of expression of particular genes. Initial changes of gene and protein expression profiles were already observed 5-8 min after colon resection. Fifteen minutes after surgery, 10%-15% of molecules, and after 30 min, 20% of all detectable genes and proteins, respectively, differed significantly from the baseline values. Significant changes of expression were found in most functional groups. As confirmed by real-time RT-PCR, this included not only known hypoxia-related molecules (HIF-1 alpha, c-fos, HO-1) but also cytoskeletal genes (e.g., CK20) and tumor-associated antigens (e.g., CEA). In conclusion, preanalytical factors, such as tissue ischemia time, dramatically affect molecular data. Control of these variables is mandatory to obtain reliable data in screening programs for molecular targets and diagnostic molecular patterns.
本研究的目的是确定缺血对健康和恶性结肠组织基因及蛋白质表达谱的影响，进而确定其对用于识别分子靶点和诊断分子模式的筛查研究的影响。在结肠切除术后的不同时间点（3 - 30分钟），将健康和恶性结肠组织速冻。分别通过微阵列（HG - U133A芯片）和表面增强激光解吸/电离飞行时间质谱技术（SELDI - TOF MS，CM10芯片、SAX2芯片和IMAC3Ni芯片）测定基因和蛋白质表达。采用实时逆转录PCR（RT - PCR）对特定基因的表达进行比较测量。在结肠切除术后5 - 8分钟即已观察到基因和蛋白质表达谱的初始变化。术后15分钟，分别有10% - 15%的分子，术后30分钟，所有可检测基因和蛋白质的20%与基线值有显著差异。在大多数功能组中均发现了显著的表达变化。实时RT - PCR证实，这不仅包括已知的缺氧相关分子（HIF - 1α、c - fos、HO - 1），还包括细胞骨架基因（如CK20）和肿瘤相关抗原（如CEA）。总之，诸如组织缺血时间等分析前因素会显著影响分子数据。在针对分子靶点和诊断分子模式的筛查项目中，控制这些变量对于获得可靠数据至关重要。
