应用功能分类基因芯片筛选卵巢癌淋巴结定向高转移细胞系中差异性表达基因

Screening and identification for lymphatic metastasis-associated differentially expressed genes in ovarian carcinoma cell lines with directional highly lymphatic metastasis using cDNA microarray

目的:在裸鼠体内建立以人卵巢浆液性乳头状腺癌细胞系SKOV3具有淋巴结定向高转移亚克隆第二代SKOV3-pm2、第三代SKOV3-pm3细胞系,应用基因芯片技术比较这3种具有不同淋巴结定向转移能力的细胞亚系中基因表达谱的差异,寻找卵巢癌侵袭转移相关基因。方法:采用Trizol一步法抽提SKOV3、SKOV3-pm2、SKOV3-pm33种细胞总RNA;取等量RNA逆转录合成cDNA用Ampolabeling-LPR(linear polymerase re-action,线性聚合酶反应)法标记的cDNA链做探针,混合后与功能分类基因芯片[OligoTumor Metastasis Microarray Catalog No.OHS-028(人)]杂交,计算机分析后比较3种细胞中差异性表达基因,并应用RT-PCR技术对其中3个表达差异明显的基因进行验证。结果:共筛选出表达差异性基因60个,其中表达上调基因47个(ratio>3),有21个基因在SKOV3-pm2、SKOV3-pm3中共同表达上调;表达下调基因13个(ratio<0.5),有1个基因在SKOV3-pm2、SKOV3-pm3细胞中共同表达下调。结论:基因表达芯片检测与肿瘤淋巴体外转移模型相结合,为肿瘤转移研究提供了新方法、新思路。

Objective：The ovarian serous papillary adenocarcinoma cell line SKOV3 and its second subclone SKOV3-pm2, the third subclone SKOV3-pm3 were instituted with directional highly lymphatic metastasis in nude mice. To screen for lymphatic metastasis-associated genes , the different gene expression profiles of the three ovarian carcinoma cell sublines with different metastatic capability were compared by cDNA microarray. Methods ：Total RNAs were isolated from cell line SKOV3,SKOV3-pm2,SKOV3-pm3 ,and reversely transcribed to the cDNA with the incorporation of fluorescent dUTP labeled by Ampolabeling-LPR after the detection of concentration and purity.9 The hybridization probes were prepared and hybridized to the cDNA microarray. Though high-stringent washing, the cDNA microarray was scanned for fluorescent signals and analyzed for different expression of the target genes between the three cell lines. Then three of the different expression genes were further validated by RT-PCR technique. Result：Among the 60 screened different expression genes,47 genes were expressed up-regulated （ ratio 〉 3 ） and 13 genes down-regulated（ ratio 〈 0.5 ）. Conclusion： Many lymphatic metas-tasis-associated genes are screened by high-throughput lar function will help to identify the key or candidate gene chip method;validating their cellu- gene/pathway responsible for lymphatic metastasis, which may be used as diagnostic marker and therapeutic targets for tumor lymphatic metastasis.