食管鳞状细胞癌组织中基因表达差异的初步分析

目的 应用寡核苷酸芯片技术分析食管鳞状细胞癌、癌旁、正常组织中基因表达差异,探讨与食管鳞状细胞癌发生、发展相关的基因变化.方法 Trizol一步法抽提食管癌、癌旁、正常组织中总RNA,纯化后逆转录为cRNA,经荧光标记、纯化,与Agilent寡核苷酸芯片(21 074探针)杂交,应用Agilent扫描仪获取图像,特征提取软件定量分析处理.挑选明显差异表达的基因进行实时荧光定量逆转录-PCR、免疫组化、免疫印迹验证.结果 ①经寡核苷酸芯片技术筛选差异表达基因的结果显示:上调38个、下调61个.②实时荧光定量逆转录PCR验证提示:差异较大的上调基因有CTHRC1、INHBA、SPP1、LUM、HRK,其中CTHRC1差异最为显著.③免疫组化:CTHRC1在食管鳞癌组织中有增强表达,表达率为56.5%(26/46).在淋巴结转移组中的阳性表达率显著高于无淋巴结转移组(P<0.05).④免疫印迹:在食管癌细胞株TE-13及Eca-109中均检测到CTHRC1的表达.结论 CTHRC1可能是食管鳞癌最为重要的生物分子之一.

Preliminary analysis of differentially expressed genes in esophageal carcinoma tissues

Objective To analyze the differentially expressed genes in esophageal squamons cell carcinoma (ESCC), para-cancerous tissue (PCT) and normal esophagus tissue (NET) using oligomicroarray and to identify the target genes related to the development and progression of esophageal carcinoma. Methods The total RNAs isolated from ESCC, PCT or NET using one step Trizol method were purified and reversely transcribed into cRNAs. The cRNAs were then fluorescence labeled and hybridized with Agilent oligomicroarray (21 074 probes). The fluorescence intensity features were detected by Agilent scanner and quantified by feature extraction software. The selected candidate genes were confirmed by real time real time fluorescent quantitative RT-PCR immunohistochemistry and Western blotting.Results ① The oligomicroarray demonstrated that there were 38 up-regulated genes and 61 down-regulated genes. ② The real time fluorescent quantitative RT-PCR revealed that five genes (CTHRC1, INHBA, SPP1 ,LUM, HRK)were more differentially expressed in up-regulated genes. Of which, CTHRC1 displayed more disparity.③ Immunohistochemistry examination showed that the higher expression of CTHRC1 (56.5 %, 26/46) was observed in ESCC. There was significantly difference in expression of CTHRC1 between patients with or without lymph node metastasis (P<0.05). ④ CTHRC1 protein was expressed in both TE-13 and Eca-109 cell lines. Conclusion CTHRC1 is probably one of the most significant biomolecules in ESCC.