细胞周期蛋白A、B1、D3、E在同步化及非同步化G_1期MOLT-4细胞中的表达

Expressions of Cyclins A, B1, D3, and E in Asynchronous and Synchronous MOLT-4 Cells of G1 Phase

背景与目的我们已经利用流式细胞术(flowcytometry,FCM)的多参数分析方法证实,用“双thymidine阻滞”获得的同步化细胞,其细胞周期蛋(Cyclins)A、B1、D3、E的表达严重失衡。但是,由于当时技术条件的限制,不可能对同期同步和非同步化的细胞中CyclinsA、B1、D3、E进行对照研究。本研究目的是用新建立的分选后的免疫印迹法(postsortingWesternblot),比较CyclinsA、B1、D3、E在同期同步及非同步化G1期MOLT鄄4细胞中的表达,证实双thymidine阻滞法诱导细胞同步化用于分析正常细胞周期的不合理性。方法以MOLT鄄4细胞为模型,用双thymidine阻滞法将细胞同步在G1/S转换期起始处的G1期,应用流式细胞术分选出同期非同步化生长的G1期细胞,采用DNA/Cyclins双参数分析法及免疫印迹法(Westernblot),检测同期同步化及非同步化G1期细胞中CyclinsA、B1、D3、E的表达。结果在分选的非同步化的G1期细胞中CyclinsA、B1几乎不表达,而在同期同步化的G1期细胞中具有明显的表达,CyclinsD3、E在同步化的G1期细胞中的表达明显高于同期分选的非同步化的G1期细胞,利用FCM的多参数分析方法和免疫印迹法所获得的结果一致。结论用双thymidine阻滞干预方法获得的同步化细胞其CyclinsA、B1、D3、E并不能代表正常细胞内的表达水平。

BACKGROUND & OBJECTIVE: We have testified expression imbalance of Cyclins A, B1, D3, and E in MOLT-4 cells, which were synchronized by 'double thymidine blocks', with flow cytometry (FCM). However, comparisons of Cyclins expressions between synchronized cells and asynchronized cells haven't been performed at that time because of technique limitations. This study was to compare expressions of Cyclins A, B1, D3, and E in G1 phase of asynchronized and synchronized cells by newly established 'postsorting Western blot', and to conform the unreasonableness of using double thymidine blocks to synchronize cells to analyze normal cell cycles. METHODS: MOLT-4 cells were synchronized at G1 phase by double thymidine blocks, asynchronous cells of G1 phase were sorted by FCM. Western blot and double parameters analysis of DNA/Cyclins were performed to detect Cyclins A, B1, D3, and E expressions in asynchronous and synchronous MOLT-4 cells of G1 phase. RESULTS: There were almost no expressions of Cyclins A, B1 in asynchronous MOLT-4 cells of G1 phase, and obvious expressions in synchronized cells of G1 phase. Expressions of Cyclins D3, E in synchronous MOLT-4 cells of G1 phase were higher than those in asynchronous cells of G1 phase. The FCM results were accordant with Western blot results. CONCLUSIONS: The expressions of Cyclins in synchronized cells obtained through 'double thymidine blocks' can't represent their expressions in normal cells. Thus, synchronous cells produced by 'double thymidine blocks' are not ideal experimental models for analyzing normal cell cycles.