腺病毒介导CD基因对人胰腺癌细胞株的体外杀伤作用

Killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro

目的 探讨腺病毒介导胞嘧啶脱氨酶基因 (CD)对人胰腺癌细胞株体外杀伤作用。方法 构建含CD基因的腺病毒穿梭载体 pAdTrack CMV CD与骨架载体 pAdEasy 1,在细菌内重组为pAd CD ,经 2 93细胞包装、扩增 ,氯化铯密度梯度离心制备纯化高效的含绿色荧光蛋白 (GFP)的CD腺病毒 ,体外转染人胰腺癌细胞株Patu 8988、SW 1990 ,并给予前药 5 氟胞嘧啶 (5 FC) ,观察其体外杀伤效果。结果 含CD基因腺病毒载体经酶切鉴定正确。包装纯化后 ,检测病毒滴度为 2× 10 11PFU/ml,将重组腺病毒转染胰腺癌细胞株Patu 8988、SW 1990后 ,可见 5 FC对转染CD基因的Patu 8988、SW 1990细胞及混育细胞有明显毒性作用 ,而对未导入CD基因的人胰腺癌细胞毒性较低。结论 体外腺病毒介导CD基因 ,不仅转染效果强 ,而且可直接或通过旁观者效应以杀伤胰腺癌细胞 。

Objective To evaluate the killing effects of cytosine deaminase (CD) gene mediated by adeno virus vector on human pancreatic cancer cell lines in vitro. Methods After CD gene was cloned into pAdTrack CMV CD, pAdTrack CMV CD and pAdEasy 1 were recombinated in bacteria. The newly recombinated Ad CD containing green fluorescent protein (GFP) was propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic cancer cell lines Patu 8988 and SW 1990 were infected with this virus, then 5 FC was added, XTT assay was used to estimate relative numbers of viable cell. Results The positive clones were selected by using endonuclease to digest the combinatants and the concentration of viral liquids containing CD gene was 2×10 11 PFU/ml. It was found that significant cytotoxic activities were possessed by 5 FC for CD gene transduced pancreatic cell lines, but little effects on the nontransduced pancreatic carcinoma cells. Conclusions CD gene mediated by adenovirus has a high infectivity and is efficient for gene therapy of pancreatic carcinoma cell lines. These data demonstrate the therapeutic efficacy of an enzyme as a prodrug strategy in experimental pancreatic cancer.