腺病毒介导的鸟氨酸脱羧酶和S-腺苷甲硫氨酸脱羧酶双反义RNA抑制肺癌A-549细胞的实验研究

Inhibitory effect of ornithine decarboxylase and S-adenosylmethionine decarboxylase bi-antisense virus on lung cancer cell A-549

目的:探讨鸟氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(AdoMetDC)双反义重组腺病毒(Ad-ODC-AdoMetDCas)介导的ODC和AdoMetDC反义RNA对肺癌A-549细胞增殖和侵袭的抑制作用。方法:采用FACS检测Ad-GFP感染细胞中GFP表达,以测定不同MOI的腺病毒对肺癌A-549细胞的基因转染效率;应用MTT法观察Ad-ODC-AdoMetDCas对肺癌A-549细胞生长增殖的影响;采用Western印迹和HPLC的方法分别检测腺病毒对肺癌A-549细胞中ODC和AdoMetDC蛋白表达以及胞内多胺含量的抑制作用,并应用TUNEL标记检测法观察Ad-ODC-AdoMetDCas对肺癌细胞凋亡的影响。结果:基因转染效率结果显示,以50 MOI的Ad-GFP感染肺癌细胞48 h后,大约有75%肺癌A-549细胞GFP表达阳性;Ad-ODC-AdoMetDCas对肺癌A-549细胞生长增殖有明显抑制作用。Ad-ODC-AdoMetDCas可明显抑制肺癌细胞中ODC和AdoMetDC基因表达,基因抑制率大于60%(P<0.05)。HPLC结果显示,肺癌A-549细胞感染Ad-ODC-AdoMetDCas后,细胞内三种多胺含量都明显降低(P<0.05)。TUNEL标记检测结果证实,Ad-ODC-AdoMetDCas可引起肺癌A-549细胞明显凋亡。结论:ODC和AdoMetDC双反义腺病毒具有显著抑制肺癌增殖和侵袭的作用,对于肺癌的防治研究具有一定的意义,有望成为治疗肺癌的基因药物。

Objective： To study the inhibitory effect of omithine decarboxylase（ODC） and S-adenosylmethionine decarboxylase bi-antisense AdoMetDCas （Ad-ODC-AdoMetDCas） virus on polyamine biosynthesis, proliferation and invasion of lung cancer cells. Methods： Adenovirus-mediated gene transduction efficiency was assessed by counting positive GFP cells using FACS. The malignant phenotype of A-549 cells was assessed by growth curves. Western Blot and HPLC were used to determine the expression of ODC and AdoMetDC and the polyamine content in A-549 cells, respectively. TUNEL was used to analyze cell apoptosis. Results： Approximate 75 % of A-549 cells were infected with Ad- ODC-AdoMetDCas when MOI reached 50. Expression of ODC was inhibited in the infected tumor cells. Ad-ODC-AdoM- etDCas inhibited growth and invasive ability of A-549 cells. TUNEL proved that Ad-ODC-AdoMetDCas could lead to cell apoptosis. Ad-ODC-AdoMetDCas vector-mediated gene transfer inhibited tumor cell growth through the blockade of the polyamine synthesis pathway. Conclusion： Ad-ODC-AdoMetDCas has a significant inhibitory effect on proliferation and invasion of lung cancer cells and has therapeutic potential for the treatment of lung cancer.