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目的构建人Smad 4基因质粒体并检测其在胶质瘤细胞系U251内的表达水平及作用。方法构建PEGFP-Smad4质粒体,应用FUGENE HD转染质粒体进入U251,G418筛选,挑出单克隆扩增,建立稳定细胞株N1-U251和Smad4-U251。将实验分为空白组(U251细胞)、N1组(空质粒-U251)、Smad4组(Smad4-U251),检测Smad4基因和蛋白表达水平;使用CCK8法检测空白组、N1组、Smad4组细胞活性。使用流式细胞术检测细胞凋亡率,使用免疫印迹技术检测Caspase 3与p-Histone H3蛋白表达水平。结果 PEGFP-Smad4质粒体构建成功,N1-U251,Smad4-U251稳定细胞株构建成功,CCK8显示空白组、N1组、Smad4组细胞活性分别是1±0.03、0.95±0.04、3.22±0.13,Smad4组与N1组比较,差异明显(P<0.05),免疫印迹显示高表达Smad 4组p Histone H 3表达水平明显增高;空白组、N1组、Smad 4组在经替莫唑胺化疗处理后细胞凋亡率分别为0.36±0.09、0.38±0.11、0.21±0.03,Smad 4组与N1组比较,差异明显(P<0.05);免疫印迹显示高表达Smad 4组Caspase 3表达水平明显降低。结论成功构建人Smad4质粒体并建立高表达Smad4的细胞系,高表达Smad4可以提高U251细胞活性,降低化疗敏感性。
Objective To construct the human Smad 4 gene plasmid and test its expression in glioma cell line U251. METHODS: PEGFP-Smad4 plasmids were constructed and transfected into U251 by FUGENE HD. The plasmids were screened by G418 and single-clonal amplification was performed to establish stable cell lines N1-U251 and Smad4-U251. The experiment was divided into blank group (U251 cells), N1 group (empty plasmid-U251), Smad4 group (Smad4-U251), detect Smad4 gene and protein expression level; CCK8 method was used to detect the blank group, N1 group, Smad4 group . The apoptosis rate was detected by flow cytometry. The expression of Caspase 3 and p-Histone H3 protein was detected by Western blotting. Results The construction of the plasmid of PEGFP-Smad4 was successful. The stable cell lines N1-U251 and Smad4-U251 were successfully constructed. CCK8 showed that the cell viability in the blank group, N1 and Smad4 groups was 1 ± 0.03,0.95 ± 0.04 and 3.22 ± 0.13, respectively Compared with N1 group, the difference was significant (P <0.05). Immunoblotting showed that the expression level of p Histone H 3 in Smad 4 overexpression group was significantly increased. The apoptosis rates of blank group, N1 group and Smad 4 group after temozolomide treatment were (0.36 ± 0.09,0.38 ± 0.11,0.21 ± 0.03). There was significant difference between Smad 4 group and N1 group (P <0.05). Western blotting showed that the expression of Caspase 3 in Smad 4 group was significantly decreased. Conclusion The human Smad4 plasmid was successfully constructed and a Smad4-overexpressing cell line was established. High expression of Smad4 could increase the activity of U251 cells and decrease the chemosensitivity.