CAJ | 학술논문

背景与目的:E2F-1(E2F transcription factor 1)基因是细胞周期的重要转录因子,也参与了细胞凋亡的过程,但机制尚不明确.本研究通过观察E2F-1过表达对胃癌细胞MGC-803凋亡的影响及对下游基因的调控,初步探讨其参与凋亡的分子机理.方法:用流式细胞仪检测稳定转染E2F-1的胃癌MGC-803/E2F-1细胞(实验组)、转染空载体的MGC-803/EV(阴性对照组)及未转染的MGC-803细胞的凋亡情况.再分别抽取MGC-803/E2F-1和MGC-803细胞的总RNA,采用逆转录的方法,制成cDNA,并以两种荧光Cy5和Cy3标记后作为探针(荧光交换芯片),与含有21 522条人类基因表达谱芯片进行杂交.采用LuxScan 10K/A双通道激光扫描仪扫描芯片上两种荧光信号,应用LuxScan3.0图像分析软件对芯片图像进行处理和分析与凋亡相关的基因的表达,再用RT-PCR针对性的对筛选得到的基因进行验证.结果:MGC-803/E2F-1组细胞的凋亡率明显高于MGC-803/EV组和MGC-803组,3组凋亡率分别为(8.40±0.91)%、(4.53±0.61)%、(4.97±0.47)%;基因芯片扫描筛选出与凋亡相关的差异表达基因15条,其中上调基因4条,下调基因11条;RT-PCR验证多条相关基因其上、下调趋势同基因芯片结果一致.结论:E2F-1基因过表达促进了胃癌MGC-803细胞的凋亡,其影响机制可能与这15条差异表达基因有关系.
배경여목적:E2F-1(E2F transcription factor 1)기인시세포주기적중요전록인자,야삼여료세포조망적과정,단궤제상불명학.본연구통과관찰E2F-1과표체대위암세포MGC-803조망적영향급대하유기인적조공,초보탐토기삼여조망적분자궤리.방법:용류식세포의검측은정전염E2F-1적위암MGC-803/E2F-1세포(실험조)、전염공재체적MGC-803/EV(음성대조조)급미전염적MGC-803세포적조망정황.재분별추취MGC-803/E2F-1화MGC-803세포적총RNA,채용역전록적방법,제성cDNA,병이량충형광Cy5화Cy3표기후작위탐침(형광교환심편),여함유21 522조인류기인표체보심편진행잡교.채용LuxScan 10K/A쌍통도격광소묘의소묘심편상량충형광신호,응용LuxScan3.0도상분석연건대심편도상진행처리화분석여조망상관적기인적표체,재용RT-PCR침대성적대사선득도적기인진행험증.결과:MGC-803/E2F-1조세포적조망솔명현고우MGC-803/EV조화MGC-803조,3조조망솔분별위(8.40±0.91)%、(4.53±0.61)%、(4.97±0.47)%;기인심편소묘사선출여조망상관적차이표체기인15조,기중상조기인4조,하조기인11조;RT-PCR험증다조상관기인기상、하조추세동기인심편결과일치.결론:E2F-1기인과표체촉진료위암MGC-803세포적조망,기영향궤제가능여저15조차이표체기인유관계.
Background and Objective: E2F transcription factor 1 (E2F-1) is an important transcription factor in cell cycle. This study was to investigate the effects of E2F-1 overexpression on apoptosis of gastric cancer MGC-803 cells and expressions of the downstream genes. Methods: The apoptotic rates were measured by flow cytometry in MGC-803/E2F-1 cells,MGC-803/EV cells or untransfected MGC-803 cells.The total RNA was extracted from MGC-803/E2F-1 cells or MGC-803 cells,and cDNA was obtained by RTPCR. Fluorescent(fluorescence exchange clip)probes marked by Cy5 and Cy3 were hybridized with gene chips containing 21522 human genes.Subsequently,the two signal images were scanned by Lux Scan 10K/A dual pathways laser scanner and analyzed by LuxScan3.0 image analysis software. RT-PCR was used to verify the target genes. Results: The apoptotic rate of MGC-803/E2F-1 cells[(8.40±0.91)%] was higher than that of MGC-803/EV [(4.53±0.61)%] and MGC-803 cells[(4.97±0.47)%].Fifteen differentially expressed apoptosis-related genes were detected,4 of which were up-expressed and 11 were down-expressed genes,and the same results were verified by RT-PCR. Conclusion: Overexpression of E2F-1 accelerates apoptosis of gastric carcinoma MGC-803 cells,which may be related to the 15 differentially expressed genes.