肿瘤转移相关基因cDNA芯片的制备与应用

Development and application of cDNA microarray of tumor metastasis-associated genes

目的建立制备肿瘤转移相关基因cDNA基因芯片的方法,并探讨其在肿瘤转移表达谱应用方面的意义。方法选择了399个已知转移相关基因克隆,经PCR扩增纯化后,以Cartesian Pixsys 5500芯片点样仪点布于多聚赖氨酸包被的玻片上;采用Cy3-dUTP和Cy5-dUTP双重荧光标记人大肠癌和肺癌细胞、正常组织、大肠癌和肺癌组织总RNA并与阵列进行杂交。结果经ScanArrayTM 4000共聚焦荧光扫描仪检测,图像背景均匀,信号清晰,效果满意。对大肠癌细胞系和组织标本及肺癌标本表达基因扫描结果行联合聚类分析发现:两种癌的基因表达大多数相同,仅少数有差别,有30种在两种转移癌中呈持续上调或下调的基因,包括运动因子基因、粘附分子、蛋白水解酶、癌基因、抑癌基因、抑制或促进凋亡基因以及诱导血管增生和信号转导的基因等。结论优化的cDNA基因芯片制备技术用于基因表达分析有较高的效率和可靠的实用性能。检测的肿瘤转移相关基因的表达在两种癌无明显差别,表明不同癌其浸润和转移的分子机制大致相同。

Objective To study the method of preparing cDNA microarray of tumor metastasis-associated genes and its application in the investigation of gene expression profile. Methods After amplification and purification by PCR, 400 tumor metastasis-associated gene clones were obtained and dotted onto the slides coated by poly-lysine. Total RNA from specimens of human colorectal carcinoma, lung carcinoma, and normal tissue samples were extracted and labeled by fluorescent staining. The labeled probes were then hybridized with the cDNA microarray. Results Uniform background and clear signal of the microarray were demonstrated by ScanArrayTM 4000. Specific mRNA expression profiles in association with colorectal cancer and lung cancer were subsequently obtained, showing that 30 genes were consistently up-regulated or down-regulated in the tissue samples examined, including motility factors, adhesion molecules, extracellular matrix-degrading enzymes, oncogenes, tumor-suppressor genes, apoptosis and anti-apoptosis genes, tumor angiogenesis factors and signal transduction factors, etc. Conclusion The optimized method is highly sensitive and applicable in examining gene expression profile. The two cancers investigated in this study do not obviously differ in light of tumor metastasis-associated gene expression profiles, indicating similar molecular mechanism accounting for the infiltration and metastatic behavior of different types of cancers.