p15^(INK4B)和p21^(WAF1)基因联合转染对人食管鳞癌细胞系EC109的协同抑制作用

Cotransfection of the p15^(INK4B) and p21^(WAF1) genes synergistically inhibits proliferation and promotes apoptosis in human esophageal squamous cell carcinoma cell line EC109

目的:探讨p15INK4B(p15)和p21WAF1(p21)基因联合转染对人食管鳞癌细胞系EC109细胞增殖和凋亡的影响.方法:脂质体介导pcDNA3.1(+)-p15和pcDNA3.1(+)-p21转染EC109细胞,稳定筛选后用RT-PCR检测转染细胞p15与p21基因mRNA表达,Western blot检测转染细胞P15和P21蛋白的表达.用MTT法和透射电镜检测p15和p21基因分别及联合转染对EC109细胞增殖与凋亡的影响,流式细胞仪检测EC109细胞周期分布与凋亡率.结果:p15和p21转染组EC109细胞生长速度低于空载体组与未转染组,联合转染组与二者单独转染组相比,亦明显抑制EC109细胞体外生长速度.p15和p21转染组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,S期则显著降低(G1期:60.52%±3.75%,63.12%±2.89%vs42.17%±5.30%,41.38%±6.54%;S期:22.67%±1.25%,17.96%±2.03%vs30.96%±3.33%,36.05%±1.78%,均P<0.01),并出现凋亡峰,透射电镜亦发现p15和p21转染组发生细胞凋亡,联合转染组发生更为明显的G1/S阻滞,G1期比例显著升高、S期比例明显降低(G1期:72.83%±2.31%vs60.52%±3.75%,63.12%±2.89%;S期:13.59%±2.59%vs22.67%±1.25%,17.96%±2.03%,均P<0.05),凋亡率明显升高(21.21%±1.78%vs4.32±1.74%,10.83%±2.40%,均P<0.01).结论:p15和p21基因联合转染在体外可以进一步增强对人食管鳞癌EC109细胞的抑制与诱导凋亡作用.

AIM： To investigate the synergistic effects of p15INK4B （p15） and p21WAF1 （p21） gene cotransfection on the proliferation and apoptosis of human esophageal squamous cell carcinoma EC109 cells. METHODS： The recombinant plasmids pcDNA3.1（＋）-p15 and pcDNA3.1（＋）-p21 were introducted into EC109 cells by liposome-mediated transfection. The expression of p15 and p21 mRNA and protein was assayed by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot, respectively. Cell proliferation was measured by methyl thiazol tetrazolium （MTT） assay. Apoptotic cells were observed using a transmission electron microscope. Cell cycle distribution and apoptosis rate were measured by flow cytometry. RESULTS： The proliferation of EC109 cells transfected with either of the two recombinant plasmids was significantly inhibited when compared with untransfected cells or cells transfected with empty plasmid. The proliferation of cotransfected cells was more significantly inhibited than that of cells transfected with either of the two recombinant plasmids. The percentages of cells in G1 phase were significantly higher in cells transfected with either of the two recombinant plasmids than in cells transfected with empty plasmid and untransfected cells （60.52% ± 3.75% and 63.12% ± 2.89% vs 42.17% ± 5.30% and 41.38% ± 6.54%, respectively; all P〉0.01）, while the percentages of cells in S phase were significantly lower in cells transfected with either of the two recombinant plasmids than in cells transfected with empty plasmid and untransfected cells （22.67% ± 1.25% and 17.96% ± 2.03% vs 30.96% ± 3.33% and 36.05% ± 1.78%, respectively; all P〉0.01）. In cotransfected cells, the percentage of cells in G1 phase significantly increased （72.83% ± 2.31% vs 60.52% ± 3.75% and63.12% ± 2.89%, respectively; both P〉0.05）, the percentage of cells in S phase significantly decreased （13.59% ± 2.59% vs 22.67% ± 1.25% and 17.96% ± 2.03%, respectively; both P〉0.05）, and apoptosis rate was significantly elevated （21.21% ± 1.78% vs 4.32% ± 1.74% and 10.83% ± 2.40%, respectively; both P〉0.01） when compared with cells transfected with either of the two recombinant plasmids. CONCLUSION： Cotransfection of the p15 and p21 genes synergistically inhibits proliferation and promotes apoptosis in EC109 cells.