摘要:
目的:研究Cyclin B1敲低后差异表达的基因,并筛选出与自噬相关的基因.方法:利用胸腺核苷(TdR)双阻断法使鼻咽癌细胞CNE-2周期同步化至S期,siRNA干扰Cyclin B1的表达,流式细胞术验证转染效率,q-PCR和western blot验证siRNA沉默效率,高通量基因芯片筛选对照组和实验组差异表达的基因.结果:经25.mmol/L的TdR同步化后,流式细胞仪检测细胞周期,获得S期细胞.用脂质体转染法将FAM-siRNA导入CNE-2细胞后,流式细胞术检测转染效率为85.6%,将Cyclin B1-siRNA导入CNE-2细胞后,Cyclin B1的mRNA和蛋白表达均较对照组明显下降85%(P<00.1).基因芯片结果显示与对照组相比,转染Cyclin B1-siRNA后一共有2408个差异表达的基因,其中1245个基因显著上调,1163个基因显著下调,初步筛选出上调的自噬相关基因PTEN.结论:Cyclin B1-siRNA可以有效地沉默Cyclin B1蛋白的表达,并可以使2408个基因差异表达,初步筛选出上调的自噬相关基因PTEN.%Objective:The study aimed to screen differentially expressed genes by silencing Cyclin B1 ,and to sift out autophagy-related genes .Methods:Double thymidine deoxyribonucleoside (TdR) blcoking was used to synchronize nasopharyn-geal carcinoma cell (CNE-2) to S phase ,then flow cytometry was applied to test transfection efficiency .The mRNA and pro-tein expression level of Cyclin B1 was assessed by q-PCR and western blot ,respectively .Differentially expressed genes were screened by high-throughput gene chip .Results:Double TdR (25. mmol/L) blocking was used to synchronize the cell cycle to S phase .The transfection efficiency of CNE-2 cells was 87% .Compared with negative group ,Cyclin B1-siRNA treated group significantly down-regulated mRNA expression of Cyclin B1 (80% ) and protein level (753.% ) (P<00.1) .Totally ,2408 differ-entially expressed genes were found in CNE-2 ,including 1245 up-regulated genes and 1163 down-regulated genes .Moreover , PTEN ,an autophagy-related gene ,was preliminarily sifted out .Conclusions :Cyclin B1-siRNA significantly down-regulated the expression of Cyclin B1 and yielded a total of 2408 differentially expressed genes ,including PETN (an autophagy-related gene) .