摘要
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(21,074探针)进行杂交,洗涤后应用Agilent Scanner获取图像,Fea-ture Extraction软件进行定量分析处理。挑选明显差异表达的基因进行SYBR GreenI染料掺入的荧光real time RT-PCR验证。结果⑴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;⑵2倍差异表达基因中,上调基因共420个,下调基因共552个,其中包括上调的基质金属蛋白酶相关基因(MMP)-9、-11和-12;⑶挑选5倍上调基因MMP-12为代表,以β-ac-tin为内对照的real time RT-PCR结果提示,MMP-12在癌、癌旁和正常肝组织中的2-ΔCt值分别为0.0030、0.0028和0.0020。结论⑴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;⑵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;⑶MMP相关基因表达上调可能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 differential expressed genes (DEGs) among hepatoeellular carcinoma(HCC), para-cancerous tissue(PCT) and normal liver tissue (NLT) using highthroughput oligomieroarray technique, explore the target genes related to the development and progression of HCC. Methods Total RNA of matched HCC, PCT and NLT tissues of HCC patients were isolated using one step Trizol method and qualified using Lab-on-chip method, eRNAs were synthesized, fluoresenee labeled and purified after total RNAs purified. The RNAs of HCC and NLT, HCC and PCT were hybrided to agilent oligomieroarray (21,074 probes) respectively. The fluorescence intensities features were detected by Agilent scanner and quantified by feature extraction software. The selected candidate genes were enntirmed by SYBR green I stained real time RT-PCR. Results (1)The total RNAs, reverse transcription products and fluoresenee labeled eRNAs were all high quality; (2)There were 420 up-regulated genes and 552 down-regulated genes in 2-fold DEGs, including 3 genes related to matrix metalloproteinases (MMPs) ; (3)MMP-12 was selected as a candidate gene of 5-fold up-regulated genes. The resuits of real time RT-PCR, using β-aetin as internal control, found that the 2-△Ct values of MMP-12 in HCC, PCT and NLT were 0.009383, 0.316812 and 0.607182, respectively. Conclusion (1)The high throughput and effective Agilent oligemicroarray can sereen novel therapy target genes by analyzing the DGEs in development and progression of HCC; (2)The development and progression of HCC is a complicated process involving muhigenes and muhiprocedures; (3)Up-regulated expression of MMP related genes maybe involved in the development and progression of HCC and the exact mechanism needs further study.
出处
《实用肿瘤学杂志》
CAS
2007年第1期23-27,共5页
Practical Oncology Journal
关键词
原发性肝癌
寡核苷酸芯片
基质金属蛋白酶
Hepatocellular carcinoma
Oligomicroarray
Matrix metalloproteinases