摘要
目的:采用基因表达系列分析(serial analysis of gene expression,SAGE)方法,分析永生化BEP2D细胞及α粒子诱发恶性转化BEP2D细胞的基因表达。方法:细胞培养,收获永生化BEP2D细胞及α粒子诱发恶性转化BEP2D细胞;提取细胞总RNA,分离mRNA,合成生物素标记的双链cDNA;采用SAGE方法获取标签序列,结合UniGene文库分析标签代表的转录本,最终通过SAGE软件分析标签丰度,比较两组细胞基因表达差异。选取SAGE实验得到的在两组细胞中表达的已知基因Smad7和 CCR11,以 RT-PCR方法制备探针,用两组细胞等量总RNA为样本,做 Northern blot杂交。结果:建立了2个独立的 SAGE文库。一个是1.5 Gyα粒子照射诱发恶性转化BEP2D细胞的SAGE文库,一个是永生化BEP2D细胞SAGE文库。从2个文库中分别挑取克隆53个、50个,进行测序,测序一共得到总标签2331个,代表单一转录本 252个,有 70%的 SAGE标签可找到与之一一对应的基因,有84个核糖体蛋白基因,约占33%;有12个转录本(4.8%)找不到与之理想匹配的已知基因;
Objective: The authors used the serial analysis of gene expression (SAGE) method to analyze transcripts present in the immortalized BEP2D cells and the malignant transformed BEP2D cells. Methods: As a step toward understanding the complex gene expression differences between the immortalized BEP2D cells and the malignant transformed BEP2D cells induced by or-particle, SAGE method was introduced into this experiment. Technological methods included total RNA extraction, mRNA isolation, full length of dscDNA synthesis, PCR, transformation and sequencing. SACE-SoftWare was used to analyze tag sequences and compare the abundance of tags between two SAGE libraries. Results: Two independent SAGE libraries were constructed from the immortalized BEP2D cells and the malignant transformed BEP2D cells induced by 1.5 Gy α-particles. A total of 2 331 SAGE tags were identified for the sequences, representing 252 unique transcripts. Though up to now the obtained information is limited, comparison of the two SAGE libraries indicated a remarkable similarity in the expression profiles. Of the 252 transcripts detected, 12 transcripts (4.8% ) matched no reliable known genes in UniGene library. Combination of Northern blot hybridization, the expression level of TGF-β induced Smad7 gene in the malignant transformed cells was higher than that in the immortalized cells. Conversely, Chemokine receptor CCR11 gene was expressed at lower levels in the malignant transformation cells. Conclusion: (1) Out of results given by SAGE, the tendency of the abundance of gene expression and the gene expression differentiation between two cell lines were described. (2) The expression level of TGF-β induced Smad7 gene in the malignant transformed BEP2D cells was higher than that in the immortalized cells and chemokine receptor CCR11 gene was expressed at lower levels in the malignant transformed BEP2D cells. (3)SAGE was a powerful method in a quantitative and simultaneous analysis of a large number of transcripts in any particular cell system, especially in defining functions of known genes.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2001年第1期12-17,共6页
Chinese Journal of Cancer
基金
国家重点基础专项经费(G1998051208)
军事医学科学院创新启动基金