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目的检测胃癌MKN45细胞周期基因表达谱的变化,从基因组学角度阐明胃癌细胞增殖的机制。方法应用双胸腺嘧啶核苷法和胸腺嘧啶核苷-偌考达唑法,分别阻断胃癌细胞株MKN45于G2／M和G1／S交界点后释放;流式细胞仪监测细胞同步化程度;cDNA基因芯片检测细胞周期中G2／M交界点、M／G1过渡期、G1早期、G1晚期、G1／S交界点、S早期、S晚期、G2早期和G2末期的基因表达谱,专业软件进行聚类分析;借助基因数据库,重点对MKN45细胞周期G1末期及G2期上调基因表达进行分析。定最PCR测定cyclin E、cyclin B、plk1、STK15基因在上述9个时间点的mRNA水平变化。结果分析基因芯片检测,得到9个时间点均可信的2001个基因,其中959个基因出现改变(上调或下调),在G1末期或G2期上调基因379个,S期和M期上调基因40个(与G1末期和G2期上凋基因重合),在G1末期上调基因中,功能主要涉及DNA代谢、转录与翻译、蛋白质转运、泛素化和信号转导等;G2期上调基因主要涉及RNA合成与加工、蛋白质转运、细胞骨架合成、凋亡与抑凋亡、转录调节、泛素化、信号转导、有丝分裂调节以及癌基因的表达等。定量PCR检测4个基因的mRNA水平变化趋势,与基因芯片检测结果基本一致。结论在MKN45细胞周期演进中,DNA复制及染色体分离所需的各种物质准备分别在G,末期及G2期完成,这种准备涉及多种类基因,成为推动MKN45细胞周期进程的主要动力,其中部分基因可能与肿瘤的过度增殖有关。基因芯片检测结果具有较好的可信度,为今后胃癌细胞周期相关基因功能研究提供了帮助。
Objective To detect the change of gene expression profile of gastric cancer cell line MKN45 and to elucidate the mechanism of gastric cancer cell proliferation from the perspective of genomics. Methods Double thymidine and thymidine - 偌 Codazol method were used to block the release of gastric cancer cell line MKN45 at the junction of G2 / M and G1 / S respectively. The cell synchronization was monitored by flow cytometry. cDNA microarray was used to detect the gene expression profiles of G2 / M junction, M / G1 transition, early G1, late G1, junction of G1 / S, S early, late S, early G2 and late G2 in the cell cycle, and professional software Cluster analysis; With the help of gene database, we focused on the analysis of up-regulated genes in G1 and G2 phases of MKN45 cell cycle. The mRNA levels of cyclin E, cyclin B, plk1 and STK15 at the above 9 time points were determined by PCR. Results Analysis of cDNA microarray revealed that there were 2001 genes that were reliable at 9 time points, of which 959 genes changed (up-regulated or down-regulated), 379 genes were up-regulated in G1 or G2 phase, 40 up-regulated genes in S phase and M phase (Coinciding with G1 and G2), up-regulating genes in G1 end mainly involved in DNA metabolism, transcription and translation, protein transport, ubiquitination and signal transduction. The up-regulated genes in G2 mainly involved RNA Synthesis and processing, protein transport, cytoskeletal synthesis, apoptosis and apoptosis, transcription regulation, ubiquitination, signal transduction, mitosis regulation and oncogene expression. Quantitative PCR detection of four genes mRNA level trends, and the results of gene chips are basically the same. Conclusions During the development of MKN45 cell cycle, various preparations for DNA replication and chromosome segregation were completed in G, terminal and G2 phases, respectively. This preparation involves multiple genes and becomes the main driving force for promoting the cycle progression of MKN45 cells. Some of them Genes may be related to the excessive proliferation of tumors. Gene chip test results have a good reliability, for the future gastric cancer cell cycle-related gene function to provide a help.