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目的了解载体携带的小干扰RNA(small interfering RNA,siRNA)对人胰腺癌细胞株PANC-1血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的抑制作用以及对肿瘤细胞生长的影响。方法设计并合成2对针对VEGF的siRNA真核表达载体(PU-VEGF-siRNA1组和PU-VEGF-siRNA2组,简称为重组质粒组),经脂质体Lipofectamine 2000转染PANC-1细胞,经G418筛选后获得稳定转染细胞株,空载体转染组作为实验对照组,未转染组作为空白对照组。采用MTT法检测转染后PANC-1细胞的增殖,流式细胞仪检测转染后PANC-1细胞的凋亡率和细胞周期,逆转录聚合酶链反应(RT-PCR)检测转染后VEGF mRNA表达情况。结果与实验对照组和空白对照组比较,两重组质粒组PANC-1细胞的增殖减慢,细胞凋亡率增加,VEGF mRNA表达明显下降,差异均有统计学意义(P<0.05);细胞周期结果显示,两重组质粒组S期细胞所占比例较两对照组明显减少(P<0.05)。结论 siRNA能有效抑制胰腺癌细胞VEGF的表达,并能在体外抑制人胰腺癌细胞株PANC-1细胞的生长。
Objective To investigate the inhibitory effect of small interfering RNA (siRNA) carried by vector on the expression of vascular endothelial growth factor (VEGF) in human pancreatic cancer cell line PANC-1 and its effect on tumor cell growth. Methods Two pairs of VEGF eukaryotic expression vectors (PU-VEGF-siRNA1 and PU-VEGF-siRNA2) were designed and synthesized. PANC-1 cells were transfected with Lipofectamine 2000, After G418 selection, stable transfected cell lines were obtained. The empty vector transfected group served as the experimental control group and the untransfected group served as the blank control group. The proliferation of PANC-1 cells was detected by MTT assay. The apoptosis rate and cell cycle of PANC-1 cells were detected by flow cytometry. The expression of VEGF was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) mRNA expression. Results Compared with the experimental control group and the blank control group, the proliferation of PANC-1 cells in both recombinant plasmids was slowed down and the apoptosis rate was increased and the expression of VEGF mRNA was significantly decreased (P <0.05). The cell cycle The results showed that the proportion of S phase cells in the two recombinant plasmids was significantly decreased compared with the two control groups (P <0.05). Conclusion siRNA can effectively inhibit the expression of VEGF in pancreatic cancer cells and inhibit the growth of human pancreatic cancer cell line PANC-1 in vitro.