在大鼠胶质瘤C6细胞中过表达Pygo2促进其细胞增殖

 目的 通过构建过表达Pygo2的重组体上调Pygo2表达，探讨其在大鼠胶质瘤C6细胞增殖中的作用及机制。方法 重组体经EcoR I和Hind Ⅲ双酶切鉴定和DNA测序后，用脂质体2000将其转染大鼠胶质瘤C6细胞，采用Western blot检测外源Pygo2蛋白表达，应用克隆形成实验和MTT法检测细胞增殖，流式细胞术检测细胞周期，采用Western blot检测过表达Pygo2对C6细胞中cyclinD1、β-catenin水平的影响，并采用细胞免疫荧光法检测其对C6细胞中cyclinD1、β-catenin亚细胞定位的影响。结果 双酶切和测序鉴定结果证实插入序列正确，重组体能有效上调Pygo2表达。将重组体转染C6细胞上调Pygo2表达后，细胞的生长增殖被显著促进，克隆形成显著增多，细胞周期进程从G1期至S期转变显著增强；且cyclinD1水平随之增高，亚定位无改变，β-catenin水平和亚细胞定位无明显改变。结论 成功构建了过表达Pygo2的重组体，过表达Pygo2通过增高cyclinD1水平，促进细胞从G1期进入S期，从而促进大鼠胶质瘤C6细胞增殖。

Over-expression of Pygo2 Promotes C6 Cells Proliferation of Glioma

Objective To up-regulate expression of Pygopus2(Pygo2) by construction of the recombinant vectors of over-expression of Pygo2 protein,and to explore the role and mechanism of over-expression of Pygo2 in C6 cells proliferation of glioma.Methods The recombinant plasmids were digested with EcoRⅠ and Hind Ⅲ to execute the restriction endonuclease identification,then the sequence analysis was assayed by DNA sequencing.The recombinant plasmids were transfected into cultured glioblastoma C6 cells using lipofectamineTM 2000.The exogenous Pygo2 protein level of C6 cells was detected by Western blot analysis.Colony forming assay and MTT assay were used to detect the cell proliferation,and cell cycle analysis was performed by flow cytometry analysis.The effect of Pygo2 over-expression on the level of cyclinD1 and β-catenin of C6 cells was detected by Western blot analysis,and the expression and subcellular location of cyclinD1 and β-catenin of C6 cells were further quantified by immunofluorescent staining.Results The recombinant plasmids were completely coincided with the designs by the restriction map and the sequence analysis,which up-regulated Pygo2 expression of C6 cells efficiently.After Pygo2 expression were up-regulated by transfected C6 cells with the recombinant plasmids,cells proliferation was promoted and colony forming was increased significantly,cell cycle progression from G1 to S transition was enhanced notably.Furthermore,the expression level of cyclinD1 was significantly increased without change of subcellular location,and the expression level and subcellular location of β-catenin were not changed obviously.Conclusion The recombinant vectors of Pygo2 over-expression were constructed successfully.Over-expression of Pygo2 promotes the growth of glioma cells by an increased expression of cyclinD1 to improve G1/S transition.