用原代细胞培养和人全基因表达谱芯片筛选鼻咽癌差异表达基因

目的 利用细胞的原代培养技术及人全基因表达谱芯片技术初步筛选出鼻咽癌差异表达基因,以发现与鼻咽癌发生、发展相关的新的候选基因.方法 分别用1例鼻咽癌细胞株C666及5例鼻咽癌培养出的原代细胞和3例鼻咽正常上皮的原代细胞,进行基因表达谱分析,并以荧光定量PCR及免疫组织化学验证芯片结果.结果 原代培养的鼻咽癌细胞和鼻咽正常上皮细胞通过细胞角蛋白的免疫细胞化学染色、EBER1原位杂交和EBV-DNA荧光定量PCR证实;鼻咽癌原代细胞和鼻咽正常上皮原代细胞的基因表达谱有显著差异;4例以上共同表达的差异基因有493个,包括上调基因264个,下调基因229个.荧光定量PCR及免疫组织化学结果和芯片结果一致.结论 用原代培养细胞及人类全基因组基因芯片构建基因表达谱能够准确、高效地筛选出鼻咽癌相关的差异表达基因,该基因表达谱的建立为研究鼻咽癌分子生物学机制、鼻咽癌的分子分型和分子诊断提供了重要的分子依据.

Identification of differentially expressed genes in primary cultured nasopharyngeal carcinoma cells by cDNA microarray

Objective To analyze the global gene expression profile of primary cultured nasopharyngeal carcinoma(NPC)cells using cDNA microarray techniques to screen new candidate genes related to the occurrence and progression of NPC.Methods A NPC cell line C666 and primary cultured NPC cells from biopsy specimens in 5 cases were analyzed with microarray techniques in comparison with 3 normal nasopharyngeal epithelial(NPE)biopsy specimens.Several differentially expressed genes identified from the microarray results were verified by fluorescence real-time PCR(FQ-PCR)and immunohistochemistry(IHC).Results Primary cultured cells of both NPC and NPE were verified by cytokeratin IHC,EBER1 in situ hybridization and EBV-DNA real-time PCR.Compared with NPE cells,a total of 493 genes in at least 4/6 of the samples were identified to be differentially expressed in the primary cultured NPC cells,including 264 up-regulated and 229 down-regulated ones.Several differentially expressed genes according to the microarray results were confirmed by real-time PCR and IHC.Conclusion cDNA microarray technique provides an effective and accurate means for global gene expression profiling of primary cultured NPC cells to screen the differentially expressed genes,which may serve as an important basis for studying the mechanism,classification and diagnosis of NPC at the molecular level.