基因芯片技术分析siRNA抑制多效生长因子表达后Pten^(-/-)MEF241细胞基因表达谱的变化  

The Change of gene expression profiles in Pten^(-/-) MEF241 cells detected by cDNA microarray

作　　者：胡迎春[1] 周乔丹[2] 张博[3] 杨柳[2] 宋博强[3] 李刚[1] 吴德昌[1] 霍艳英[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850 [2]重庆医科大学病理学教研室,重庆400016 [3]军事医学科学院附属307医院,北京100071

出　　处：《军事医学科学院院刊》2007年第5期405-410,共6页Bulletin of the Academy of Military Medical Sciences

基　　金：国家自然科学基金项目(No.30471946);北京市自然科学基金项目(No.7052056)

摘　　要：目的:应用基因芯片技术,分析siRNA抑制Pleiotrophin基因表达后,Pten-/-MEF241细胞基因表达谱的变化。方法:选用Agilent公司的小鼠oligo芯片,分别提取siRNA抑制Pleiotrophin基因表达前后Pten-/-MEF241细胞的RNA,反转成cDNA,进一步荧光标记后进行芯片杂交,杂交结果经扫描及软件分析,最后Ration值为cy3/cy5(即实验组/对照组)。差异基因筛选标准为正标Ratio(Cy3/Cy5)≥2同时反标Ratio(Cy3/Cy5)≤0.5。部分上调及下调基因的表达水平用RT-PCR及Northern杂交进行了验证。结果:siRNA介导Pleiotrophin基因沉默后,表达上调2倍以上的基因有240个,下调0.5倍以上的基因有129个。其中,上调10倍以上的基因有与DDK综合征相关的Schlafen家族成员Slfn2、3、4,基质蛋白金属酶Mmp3、10、13,白介素IL-1a、IL-f6等;下调5倍以上的基因有钙结合蛋白S100a8、视黄醇结合蛋白Rbp4、二肽酶Dpep1等。RT-PCR及Northern杂交验证结果与芯片结果吻合。结论:应用基因表达谱芯片成功分析了RNAi抑制Pleiotrophin基因表达后Pten-/-MEF241细胞基因表达谱的变化,挑选并鉴定出一批有意义的基因,为后续的深入研究提供了基础。Objective ：To explore the change of gene expression profiles in Pten^ -/- MEF241 cells, in which the secretion of growth factor pleiotrophin（Ptn） was inhibited by the Ptn-specific siRNA and normal Pten^-/- MEF241 cells using cDNA microarray. Methods： The total RNAs were isolated from the Ptn-inhibited Pten-^/- MEF241 cells and normal Pten ^-/- MEF241 cells. Total RNAs were purified and subjected to reverse transcription into first-strand cDNA. The cDNA probes were prepared by labeling with Cy3 and Cy5 fluorescence dyes individually and hybridized to the Agilent rat oligo microarray containing approximately 22392 rat genes. Differential expression genes were analyzed using Aglient Feature Extraction software. Results： Expression of 240 genes was up-regulated by two-fold in the Ptn-inhibited Pten^-/- MEF241 cells, while expression of 129 genes was clown-regulated by 0.5 time. Conclusion：The difference of gene expression profiles between the Ptn-inhibied Pten ^-/- MEF241 cells and normal Pten^-/- MEF241 cells was suceessfully analyzed by cDNA microarray.

关 键 词：寡核苷酸序列分析 基因表达 基质金属蛋白酶 逆转录聚合酶链反应 

分 类 号：Q78[生物学—分子生物学]