p16^(INK4α)重表达对人食管癌细胞系EC109生长的抑制作用

Re-expression of p16^(INK4α) gene suppresses growth of human esophageal carcinoma cells

目的探讨用腺病毒介导野生型的 p16基因在食管癌细胞系表达,研究重表达野生型 p16基凶对食管癌细胞系 EC109生长的抑制作用,为寻找食管癌致癌机制的研究及其基因治疗提供理论基础.方法用基因重组技术,将 pcDNA3-p16中的野生型 p16基因用 Kpn I/Bam H (?)进行双酶切,克隆入腺病毒表达载体pAdCMV 中,将重组质粒 pad-CMV-p16与腺病毒质粒 JM17用Lipofectamime 2000共转染293细胞,产生重组腺病毒.用重组腺病毒感染人食管癌细胞系 EC109,在感染后的不同时段,用免疫荧光、打点杂交方法检测 p16基因在细胞中的表达.用MTT 法及流式细胞仪观察 p16基因的表达对食管癌细胞株生长的影响.结果经酶切鉴定野生型 p16基因克隆入腺病毒表达载体中,与 JM17共转染293细胞后,可产生具有感染活性的重组腺病毒,用重组腺病毒感染食管癌细胞株 EC109细胞后,可抑制食管癌细胞的生长,同对照组相比其最大抑制率可达52.7%.Multipcycle 分析软件分析对细胞周期影响表明,处于 G_0～G_1期的细胞为41%～63%.感染后的细胞经 Dot blot 和 Westernblot 杂交证实,有外源的 p16mRNA 及蛋白的表达.结论重组腺病毒可将野生型 p16基因导入人食管癌细胞系EC109中,野生型 p16基因在 p16基因功能缺失的细胞中重表达能抑制癌细胞恶性生长.p16基因功能缺失是食管癌致癌因素之一.

AIM To study the wild-type p16cDNA expressed in the esophageal carcinoma cells by the adenovirus expression vector,to detect the effect of p16 gene on the growth inhibition of esophageal carcinoma cell line EC109,and provide a background of gene therapy for esophageal carcinoma. METHODS The wild-type p16cDNA was cut from pcDNA3- p16 by Kpn[/BamH I and cloned into the adenovirus expression vector pAdCMV,pad-CMV-p16 and JM17 were transfected into 293 cells which produced the recombined adenovirus.Using the recombined adenovirus to infect the EC109 cell,the p16 gene and protein were detected with Dot blot hybridization and immunofluorescence technique. The effect of p16 was observed on the growth inhibition by MTT and FCM. RESULTS p16 gene was cloned into the adenovirus expression vector pAdCMV.It can produce the recombined adenovirus in the 293 cells.After the EC109 cells were infected by the recombined adenovirus, immunofluorescence technique and Dot blot hybridization sbowed increased level of p16 expression.The progression of cell cycle was arrested from G_1 to S phase. CONCLUSION Transduction of wild type p16 gene into p16 gene-depleted esophageal carcinoma cells can restore its suppressive effect on cell growth by arrest of cell cycle at G_1 phase.