抑癌PTEN基因联合p53基因转染对前列腺癌PC-3m细胞凋亡的影响

Effect of combined cancer-suppressing PTEN and p53 gene transfection on apoptosis of prostate cancer PC-3m cells

目的研究抑癌PTEN基因联合p53基因转染对前列腺癌PC-3m细胞凋亡的影响。方法构建表达载体:空质粒pEGFP-N1、pEGFP-N1-PTEN质粒、pEGFP-N1-p53质粒及pEGFP-N1-PTEN-p53质粒,体外分别转染到前列腺癌PC-3m细胞中,观察它们转染后的荧光表达情况;采用RT-PCR法检测目的基因表达;Western blotting法检测目的 PTEN蛋白和P53蛋白的表达;用MTT法观察细胞增殖抑制情况并绘制生长抑制曲线;Annexin V-FITC/PI双染流式细胞术检测PTEN基因和p53基因对前列腺癌PC-3m细胞凋亡的影响。结果荧光显微镜下观察到PTEN、p53及两个基因同时在PC-3m细胞系中成功表达;RT-PCR检测结果显示,与PTEN基因单转染组、p53基因单转染组和空载体转染组相比联合转染组mRNA在PC-3m细胞系中表达明显增加(P<0.05);Western blotting法检测同样发现,联合转染组PTEN蛋白及P53蛋白表达明显高于PTEN基因单转染组、p53基因单转染组和空载体转染组(P<0.05);联合转染组细胞凋亡率明显高于空载体转染组、PTEN基因单转染组和p53基因单转染组(P<0.05)。结论 PTEN基因与p53基因联合转染能诱导前列腺癌PC-3m细胞凋亡,这种联合转染可能对前列腺癌的基因治疗具有潜在的应用价值。

Objective To study the effect of combined cancer-suppressing PTEN and p53 gene on apoptosis of prostate cancer PC- 3m cells. Methods Expression vector was constructed for empty plasmid pEGFP-N1, pEGFP-N1-PTEN plasmid, pEGFP-N1- p53 plasmid and pEGFP- N1-PTEN-p53 plasmid, respectively. Their fluorescence expression was observed under microscope after transfection. Expressions of target gene, PTEN and p53 protein were detected by RT-PCR and Western blot, respectively. Cell proliferation inhibition was assayed by MTT assay and cell growth curve was plotted. Effect of PTEN and p53 gene on apoptosis of prostate cancer PC-3m cells was detected by Annexin V-FITC/PI cytometry. Results Fluorescence microscopy showed that PTEN and p53 gene were expressed in PC-3m cells. RT-PCR and Western blot displayed that the expression levels of PTEN and p53 mRNA were significantly higher in combined PTEN and p53 gene-transfected PC-3m cells than in PTEN-, p53- and empty vector- transfected PC-3m cells （P 〈 0.05）. The apoptosis rate of combined PTEN and p53 gene-transfected PC-3m cells was significantly higher than that of PTEN-, p53- and empty vector-transfected PC-3m cells （P 〈 0.05）. Conclusion Transfection of combined PTEN and p53 gene can induce apoptosis of prostate cancer PC-3m cells and can thus be applied in gene therapy for prostate cancer.