p15^(INK4B)基因转染对人食管鳞癌细胞EC109增殖的抑制作用

Inhibitory effect of p15^(INK4B) gene transfection on proliferation of human esophageal squamous cell carcinoma cell line EC109

目的:探讨p15INK4B(p15)基因转染对食管鳞癌细胞系EC109细胞增殖的影响.方法:根据转染质粒的不同和是否进行质粒转染分为3组:p15转染组,空载体转染组,未转染组.应用PCR检测外源性p15基因,Westernblot方法检测转染细胞的P15蛋白变化:流式细胞仪分析细胞周期变化,应用MTT、集落形成实验、流式细胞仪和透射电镜检测转染外源p15基因对EC109细胞增殖和凋亡的影响.结果:p15转染细胞存在外源p15基因,并有P15蛋白高表达;EC109-p15细胞生长速度低于对照细胞EC109-空载体组和未转染组,集落形成率显著低于对照细胞EC109-空载体组和未转染组(20.8±1.3%vs54.3±3.2%,56.8±2.3%,P<0.01);流式细胞仪观察到P15蛋白高表达使EC109细胞发生G1/S阻滞,G1期细胞比例显著高于空载体组和未转染组(62.4±7.1%vs38.0±5.8%,34.4±1.0%,P<0.01),S期比例显著低于空载体组和未转染组(21.1±1.3%vs35.5±2.4%,36.3±0.7%,P<0.01),并出现亚G1峰(凋亡峰).透射电镜亦发现p15转染组发生细胞凋亡.结论:p15基因转染可以抑制人食管鳞癌细胞系EC109细胞增殖并能诱导其发生细胞凋亡.

AIM： To investigate the effect of p15^INK4B （p15） gene transfection on the proliferation of esophageal squamous cell carcinoma cell line EC109. METHODS： The EC109 cells were divided into 3 groups： p15 transfection group, empty plasmid transfection group and non-transfection group, pCDNA3.1 （＋）-p15 and pCDNA3.1 （＋）-neo plasmid were transfected into EC109 cells using Lipofectamine2000. The expression of exogenous p15 gene and P15 protein were detected by polymerase chain reaction （PCR） and western blot, respectively. The proliferation and apoptosis of the EC109 cells wereexamined by MTT, colony formation assay, flow cytometry and transmission electron microscope. RESULTS： After transfection, p15 gene cDNA was expressed in the cells of p15 transfection group, and P15 protein was expressed at a high level. The cell growth was inhibited, and the colony formation was significantly decreased in EC109-p15 cells as compared with that in the cells of empty plasmid transfection or non-transfection group （20.8±1.3% vs 54.3 ± 3.2%, 56.8 ± 2.3%, P〈0.01 ）. EC109-p15 cells arrested at G1/S phase, and the population of the cells in G1 phase was significantly increased as compared with that in the cells of empty plasmid transfection group or Non-transfection g r o u p （62.45 ± 7.08% vs 38.02 ± 5.83%, 34.45 ± 1.05%, P〈0.01）. However, the number of the cells in S phase was significantly decreased （21.12 ± 1.31 % vs 35.50 ± 2.38%, 36.30± 0.69%, P〈0.01）, and a Sub G1 peak （apoptosis peak） appeared. Under electron microscope, typical features of apoptosis were observed in EC109-p15 cells. CONCLUSION： p15 gene transfection can not only inhibit the proliferation of EC109 cells, but also induce the apoptosis of the cells in vitro.