p15^(INK4B)和p21^(WAF1)基因对人胰腺癌细胞系BxPC3的协同抑制作用

Synergistic inhibitory effect of p15^(INK4B) and p21^(WAF1) gene on human pancreatic cancer cell line BxPC3

目的探讨p15INK4B(p15)和p21WAF1(p21)基因联合转染对人胰腺癌细胞系BxPC3细胞增殖和凋亡的影响。方法脂质体介导将pcDNA3.1(+)-p15和pcDNA3.1(+)-p21转染BxPC3细胞,稳定筛选后用RT-PCR检测转染细胞p15和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BxPC3细胞增殖和凋亡的影响,流式细胞仪检测BxPC3细胞周期分布和凋亡率。结果 p15和p21转染组BxPC3细胞生长速度低于空载体组和未转染组,联合转染与二者单独转染相比,细胞体外生长速度受抑制更明显。p15和p21转染组BxPC3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期比例显著低于空载体组和未转染组,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。p15和p21联合转染组与二者单独转染相比,发生更为明显G1/S阻滞,G1期比例升高,有统计学意义,S期比例降低,但无统计学意义,凋亡率升高,有统计学意义。透射电镜发现联合转染组诱导BxPC3发生更明显的细胞凋亡。结论 p15和p21基因联合转染可以进一步增强对人胰腺癌BxPC3细胞的抑制作用和诱导凋亡作用。

Objective To investigate the effect of pl5INK4B（plS） and p21WAVl（p21 ）gene co- transfection on proliferation and apoptosis of human pancreatic cancer cell line BxPC3. Meth- ods The plasmid pcDNA3.1 （ ＋ ）-p15 and peDNA3.1 （ ＋ ）-p21 were introdueted into BxPC3 cells by the vector of liposomes. The p15 and p21 gene mRNA and protein expression were as- sayed by RT-PCR and Western blot. The proliferation and apoptosis were studied by means of M3~I＇ assay and transmission electron microscope. Cell cycle and population of apoptosis were measured by flow cytometry. Results After p15 and p21 gene cotransfection, the expression of the two genes mRNA and protein was at high level. The BxPC3 cells growth was significantly decreased in p15 or p21 gene transfection group compared with that either in empty plasmid transfection group or h3. nontransfection group. The growth of cotransfection group cells was sig- nificantly decreased compared with that in either p15 transfection group or p21 transfection group. In the cotransfection group, the population of Gi phase cells was significantly increased, and the population of the apoptosis cells was significantly increased, compared with that in ei- ther.pl5 or p21 transfection group, but the decrease of S phase cells had no statistical significance. There was also found more obvious apoptosis by transmission electron microscope in the cotransfection group. Conclusion p15 and p21 gene cotransfection could enhance prolifera- tion inhibition and induce more apoptosis on BxPC3 cells in vitro.