双反义腺病毒载体Ad-ODC-AdoMetDCas对食管癌细胞凋亡及抑制细胞生长影响的研究

The Effect of Double Antisense Virus Ad-ODC-AdoMetDCas on Apoptosis and Growth of Eca109 Esophageal Cancer Cells

目的:探讨双反义腺病毒载体(Ad-ODC-AdoMetDCas)对食管癌Eca109细胞凋亡作用及抑制细胞生长的影响。方法:应用MTT法测定不同MOI的腺病毒对肺癌Eca109细胞的基因转染效率,观察Ad-ODC-AdoMetDCas对食管癌Eca109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载体对食管癌Eca109细胞中ODC和AdoMetDC蛋白表达以及胞内多胺含量的抑制作用,采用流式细胞术检测腺病毒介导的鸟氨酸脱羧酶反义RNA(Ad-ODCas)和Ad-ODC-AdoMetDCas对食管癌Eca109细胞周期分布的影响,同时应用原位末端标记(TUNEL)法观察Ad-ODC-AdoMetDCas对食管癌Eca109细胞凋亡作用的影响。结果:MTT法实验表明,Ad-ODC-AdoMetDCas对食管癌Eca109细胞生长增殖有显著抑制作用(P<0.05)。以Ad-ODC-AdoMetDCas感染食管癌Eca109细胞,可明显抑制食管癌Eca109细胞中ODC和AdoMetDC基因表达(P<0.05)。流式细胞术结果显示感染了Ad-ODC-AdoMetDCas的食管癌Eca109细胞周期阻滞在G1期。HPLC结果显示,食管癌Eca109细胞感染Ad-ODC-AdoMetDCas后,细胞内3种多胺含量均明显降低(P<0.05)。TUNEL标记检测结果显示,Ad-ODC-AdoMet-DCas可明显引起食管癌Eca109细胞凋亡(P<0.05)。结论:Ad-ODC-AdoMetDCas能显著抑制食管癌细胞生长,促进细胞凋亡,为探讨食管癌基因治疗的可行性提供实验依据。

Objective： To study the inhibitory effects of Ad-ODC-AdoMetDCas on polyamine biosynthesis, growth and apoptosis of esophageal cancer cells. Methods： Adenovirus-mediated gene transduction efficiency was assessed by counting GFP-positive cells using MTT. The malignant phenotype of Eca109 cells was assessed by growth curve. Western blot and HPLC were used to detect ODC and AdoMetDC expression and polyamine content in Eca109 cells. Viable cell cycle analysis was employed to evaluate cell cycle distribution. TUNEL was used to assess apoptosis. Results： Approximate 70% of Eca109 cells were infected with Ad-ODC-AdoMetDCas when an MOI of 50 was used. The expression of ODC was inhibited in the infected tumor cells. Ad-ODC-AdoMetDCas inhibited Eca109 cell growth and invasive ability. The tumor cells were arrested in G1 phase after gene transfer. TUNEL analysis showed that Ad-ODC-AdoMetDCas induced cell apoptosis. Conclusion： Ad-ODC-AdoMetDCas has significant inhibitory effects on esophageal cancer cell proliferation and thus has a therapeutic potential for the treatment of esophageal cancer.