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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 ,可作为胰腺癌基因治疗的有效方法
Objective To investigate the in vitro cytotoxicity of adenovirus-mediated cytosine deaminase (CD) on human pancreatic cancer cell lines. Methods The recombinant adenoviral shuttle vector pAdTrack CMV CD containing CD gene and its backbone vector pAdEasy 1 were constructed and recombined into pAd CD in bacteria. The recombinant adenovirus was then packaged into 293 cells for amplification and cesium chloride density gradient centrifugation to prepare purified and highly efficient green fluorescent protein (GFP) CD adenovirus, in vitro transfection of human pancreatic cancer cell line Patu 8988, SW 1990, and given prodrug 5 flucytosine (5 FC) to observe its in vitro killing effect. Results The recombinant adenoviral vector containing CD gene was identified by restriction enzyme digestion. After packaging and purification, the virus titer was determined to be 2 × 10 11 PFU / ml. After the recombinant adenovirus was transfected into the pancreatic cancer cell line Patu 8988 and SW 1990, 5 FC was transfected into Patu 8988 and SW 1990 cells transfected with CD gene and mixed Fertile cells have a significant toxic effect, while the non-introduction of CD genes in human pancreatic cancer cells are less toxic. Conclusion In vitro adenovirus mediated CD gene not only has strong transfection efficiency, but also can kill pancreatic cancer cells directly or bystander effect and can be used as an effective method for gene therapy of pancreatic cancer