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目的应用寡核苷酸芯片技术高通量分析原发性肝癌与癌旁组织以及正常肝组织的基因表达差异,探索与疾病进展相关的靶基因。方法抽提原发性肝癌患者的癌组织、癌旁组织以及正常肝组织的总RNA,应用芯片实验室(Lab-on-chip)进行RNA质量检测;总 RNA纯化后进行逆转录cRNA合成、荧光标记和纯化,将癌组织和正常肝组织、癌组织和癌旁肝组织的cRNA探针分别与Agilent寡核苷酸芯片(21074探针)进行杂交,洗涤后应用Agilent Scanner获取图像,Feature Extraction软件进行定量分析处理。挑选在癌和癌旁以及癌与正常组织中均明显差异表达的基因进行SYBR Green Ⅰ染料掺入的荧光real time RT-PCR验证。结果①癌组织、癌旁肝组织以及正常肝组织的总RNA质量高,反转录cRNA及荧光标记质量好;②2倍差异表达基因中,上调基因共420个,下调基因共552个,其中包括细胞色素P450相关基因23个;③挑选10倍下调基因CYP2C8为代表,以β-actin为内对照的real time RT-PCR结果提示,CYP2C8在癌、癌旁和正常肝组织中的2-ΔCt值分别为0．009 383、0．316 812和0．607 182。结论①应用Agilent寡核苷酸芯片高通量、高效率地分析原发性肝癌发生发展过程中的基因表达差异,可以筛选新的治疗靶点; ②原发性肝癌的发生发展涉及多基因、多步骤,是一个复杂的过程;③细胞色素P450相关基因表达下降可能导致致癌物的活性增加,涉及原发性肝癌的发生发展过程,确切机制有待深入研究。
OBJECTIVE: To analyze the differences of gene expression between primary hepatocellular carcinoma (HCC) and normal liver tissues by using oligonucleotide microarray technique and explore the target genes related to disease progression. Methods Total RNA was extracted from patients with primary liver cancer and normal liver tissues by using a Lab-on-chip method. Total RNA was purified and reverse-transcribed cRNA was synthesized. Fluorescence After labeling and purification, the cRNA probes of cancer tissues, normal liver tissues, cancer tissues and paracancer liver tissues were respectively hybridized with the Agilent oligonucleotide chip (21074 probe), and the images were scanned with an Agilent Scanner after washing. The Feature Extraction software Quantitative analysis and processing. Fluorescence real time RT-PCR was performed to identify genes that were significantly differentially expressed in cancer and adjacent tissues as well as in cancer and normal tissues. Results ① The total RNA quality of cancer tissue, adjacent liver tissue and normal liver tissue were high, and the quality of reverse transcription cRNA and fluorescent marker was good. Among the 2-fold differentially expressed genes, there were 420 up-regulated genes and 552 down-regulated genes, including Cytochrome P450-related genes 23; ③ select 10 times down-regulated gene CYP2C8 as a representative, β-actin as an internal control real time RT-PCR results suggest that CYP2C8 in cancer, adjacent and normal liver tissue 2-ΔCt value Respectively 0.009 383,0.316 812 and 0.607 182. Conclusion ①Using Agilent oligonucleotide microarray to analyze the gene expression differences in the development and progression of primary hepatocellular carcinoma (HCC) with high-throughput and high-efficiency, and to screen for new therapeutic targets. The occurrence and development of primary HCC involve multiple genes, Multi-step, is a complex process; ③ cytochrome P450 related gene expression decline may lead to increased activity of carcinogens, involving the development of primary liver cancer, the exact mechanism needs further study.