Akt1和Akt2基因转染对人胃黏膜上皮细胞GES-1生长的影响

目的观察Akt1和Akt2基因转染人胃黏膜上皮GES-1细胞后对其生长的影响。方法采用脂质体法将分别含Akt1和Akt2基因全长cDNA序列的p-LXSN-Akt1(Akt1组)和p-LXSN-Akt2(Akt2组)质粒转染人胃黏膜上皮GES-1细胞,以转染p-LXSN空质粒的细胞作为空载组,正常未转染细胞作为对照组,并经抗生素G418筛选抗性克隆。提取各组细胞的总蛋白,蛋白质印迹法检测转染后细胞Akt1、Akt2蛋白及细胞周期蛋白D1(CyclinD1)的表达水平;采用MTT法检测转染后细胞的增殖活性;应用流式细胞仪检测Akt1和Akt2基因转染对细胞周期的影响。结果 Akt1和Akt2组均获得了稳定表达Akt1和Akt2基因的GES-1细胞模型。Akt1组:①Akt1蛋白相对表达量(0.96±0.05)分别是空载组(0.47±0.02)和对照组(0.64±0.03)的2.03±0.22和1.48±0.19倍(均P0.01),CyclinD1蛋白相对表达量(0.74±0.01)分别是空载组(0.29±0.01)和对照组(0.22±0.01)的2.57±0.05和3.44±0.04倍(均P0.01);②吸光度(A490)值于第1、2天分别与空载组和对照组比较差异无统计学意义,第3～6天差异均有统计学意义(均P0.01);③S期细胞数与空载组和对照组比较分别增多约20.00%和21.10%,G2期细胞数减少约20.80%和15.48%(均P0.01)。Akt2组:①Akt2蛋白相对表达量(0.95±0.01)分别是空载组(0.62±0.02)和对照组(0.56±0.01)的1.53±0.04和1.69±0.01倍(均P0.01),CyclinD1蛋白相对表达量(0.31±0.02)与空载组(0.29±0.01)和对照组(0.22±0.01)比较差异均无统计学意义;②A490值于第1～6天分别与空载组和对照组比较差别均无统计学意义;③S期细胞数与空载组和对照组比较差异均无统计学意义。结论 Akt1基因可使GES-1细胞S期细胞数增多,并提高细胞的增殖能力;而Akt2基因对GES-1细胞的细胞周期和增殖能力的影响不明显。

The effect of Akt1 and Akt2 gene transfection on growth of human gastric epithelial cell GES-1

Objective To observe the effects of Akt1 and Akt2 genes transfection on the growth of human gastric epithelial GES-1 cells. Methods The plasmids of p-LXSN-Akt1 which contained the whole Akt1 gene sequence (Akt1 group) and p-LXSN-Akt2 which contained the whole Akt2 gene sequence (Akt2 group) were used to transfect human gastric epithelial GES-1 cells with lipofectamine, the GES-1 cells which were transfected by the empty vector p-LXSN were used as the black vector group, and the no transfected ones were used as the control group. Then the positive cell clones were selected with antibiotics G418. The protein expressions of Akt1, Akt2 and Cyclin D1 in the GES-1 cells were measured by Western blotting analysis; MTT method was applied to detect the proliferation activity of GES-1 cells; the effect of Akt1 and Akt2 genes transfection on the cells cycle of the GES-1 cells was tested by flow cytometry (FCM). Results Cell modes which expressed Akt1 gene (Akt1 group) and which expressed Akt2 gene (Akt2 group) were obtained. Akt1 group: ①The relative expressions of Akt1 protein (0.96 ± 0.05) were respectively 2.03 ± 0.22 times of the black vector group (0.47 ± 0.02) and 1.48 ± 0.19 times of the control group (0.64 ± 0.03) (all P < 0.01), the relative expressions of Cyclin D1 protein (0.74 ± 0.01) were respectively 2.57 ± 0.05 times of black vector group (0.29 ± 0.01) and 3.44 ± 0.04 times of the control group (0.22 ± 0.01) (all P < 0.01); ②Compared with the black vector group and control group, the absorbance (A490) values in the first 1, 2 days were no significant differences, but there were statistically significant differences (all P < 0.01) in the first 3 to 6 days; ③Compared with the black vector group and control group, S phase cells were increased by approximately 20.00% and 21.10%, G2 phase cells were reduced by approximately 20.80% and 15.48% (all P < 0.01). Akt2 group: ①The relative expression of Akt2 protein (0.95 ± 0.01) were 1.53 ± 0.04 times of the black vector group (0.62 ± 0.02) and 1.69 ± 0.01 times of the control group (0.56 ± 0.01) (all P < 0.01), compared with the black vector group (0.29 ± 0.01) and the control group (0.22 ± 0.01), the relative expression of Cyclin D1 protein (0.31 ± 0.02) was no significant differences; ②Compared with the black vector group and the control group, the A490 values of the first 1 to 6 days were no significant differences; ③Compared with the black vector group and control group, S phase cells were no significant differences. Conclusion Akt1 gene transfection could increase the proportion of S-phase cells and enhance the proliferation ability of the GES-1 cells, but the effects of Akt2 gene was not obvious.