突变型Axin2对细胞增殖的影响及其机制的研究

目的Axin是新近发现的Wnt信号传导系统的抑癌基因，本文在体研究其羧基端缺失突变（mtAxin2）对细胞增殖的影响并探索其相关机制。方法采用pCMV-Flag—mtAxin2质粒瞬时转染293细胞，并用G418筛选稳定表达mtAxin2的细胞株；使用Dual—Luciferase报告检测系统检测Firefly和Renilla荧光酶活性，采用TrpanBlue染色方法检测细胞增值活性，用流式细胞仪检测mtAxin2对细胞周期的影响，应用Western blot方法检测mtAxin2对Wnt信号传导系统下游基因的影响，G0／1细胞同步化S期进入实验研究mtAxin2对细胞生长影响的机理。结果经G418筛选后在293细胞中获得的细胞株，westem blot检测可见明显的mtAxin2蛋白表达；Western blot和Dual—Luciferase报告检测系统显示在稳定表达mtAxin2的细胞株中β-catenin蛋白水平增加，TCF活性增高；通过Trypan Blue排除实验计数绘制细胞生长曲线，显示mtAxin2促进细胞的生长；流式细胞仪检测发现在稳定表达mtAxin2的细胞株中S期细胞的比例较高。Western blot结果显示在稳定表达mtAxin2的细胞株中β-catenin蛋白水平上升的同时，磷酸化的β-catenin蛋白水平却下降，下游基因产物CyclinDl和cdc2表达也增加；G0／G1期同步化细胞释放后12h流式细胞仪检测发现稳定表达mtAxin2的细胞株中S期细胞比例明显增加，为293细胞的2—3倍。结论Wnt信号传导系统在细胞生长中起重要作用，mtAxin2通过cyclinD1促进了细胞的增殖，发挥癌基因的作用。

Mechanism and influence of a truncated mutation of Axin2 on cell proliferation

Objective Axin2 gene, a negative regulator of Wnt signaling, was cloned recently. The present study was to explore the in vivo effects of a truncated mutation of Axin2 (mtAxin2) on cell proliferation and its relevant mechanism. Methods wtAXIN2 and mtAXIN2 plasmids were prepared for TCF luciferase activity assay and stable cell lines of mtAXIN2 selected with G418. Firefly and Renilla activities of mtAXIN2 were detected with Dual-Luciferase Report Assay System. Cell proliferation curve, colony formation assay and flow cytometry were used for mtAXIN2 stably expressed cells to detect its effects on cell proliferation and cell cycle. Minosine was used for cell synchronization and S phase entry assay. Results In G418-selected mtAxin2 stable cell lines, western blot showed the expression of exotic Flag-tagged mtAxin2 and TCF activity increased in these cell lines in Dual-Luciferase Report assay. With trypan blue staining, we plotted the cell proliferation curves and found that cell growth was moderately promoted in the mtAxin2 stable cell lines. Flow cytometry assay showed that the proportion of cells in S phase increased in the mtAxin2 stable cell lines. Western blot showed that the levels of &;#61538;-catenin, cyclinD1 and cdc2 protein increased, but the phosphorylated &;#61538;-catenin level reduced in the mtAxin2 stable cell lines. Flow cytometry assay showed that the proportion of S phase cells increased dramatically in the mtAxin2 stable cell lines 12hr after release from G0/1 synchronization. Conclusion mtAxin2 could promote the in vivo cell proliferation via cyclin D1 pathway.