Adenoviral mediated suicide gene transfer in the treatment of pancreatic cancer

腺病毒介导自杀基因治疗胰腺癌实验研究(英文)

OBJECTIVE: To determine the efficacy of adenovirus mediated suicide gene transduction combined with prodrug 5-fluorocytosine (5FC) as a therapeutic protocol for pancreatic cancer. METHODS: Cytosine Deaminase(CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria. The newly recombined adenovirus (Ad)-CD containing green fluorescent protein (GFP) were packaged and propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell line-Patu8988 was infected with this virus, then 5FC was added. XTT assay was used to estimate relative numbers of viable cells. In vivo model of pancreatic cancer was established by injecting 1.0 x 10(7) Patu8988 cells subcutaneously in Balb/c nude mice. When tumors were palpable, Ad-CD was injected into each tumor and 5FC was administered. RESULTS: Positive clones were selected using endonuclease to digest the recombinants and the concentration of viral liquids containing the CD gene was 2 x 10(11) pfu /ml. Significant cytotoxic activity as shown for 5FC in the CD gene transduced 8988 cell line, while little effect was found in the nontransduced pancreatic carcinoma cells. Antitumor effect was observed in Patu8988 xenograft nude mice with in situ CD gene transduction. CONCLUSIONS: CD gene mediated by adenovirus has high infectivity and may be useful for gene therapy in pancreatic carcinoma. These data demonstrate the use of an enzyme prodrug strategy in experimental pancreatic cancer.

目的 探讨腺病毒介导自杀基因联合前药 5FC对人胰腺癌的治疗作用。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在细菌内同源重组为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greenfluorescentprotein (GFP)的CD腺病毒 ,体外转染人胰腺癌细胞Patu8988,并给予前药 5 FC ,观察其体外杀伤效果 ,Balb/c裸鼠皮下注射 1 0×10 7个人胰腺癌细胞Patu8988制成体内胰腺癌模型 ,待成瘤后 ,CD腺病毒直接注射至肿瘤 ,并腹腔给予 5FC。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Patu8988后 ,可见 5FC对转染CD基因的Patu8988细胞及混育细胞有明显细胞毒性作用 ,而对未导入CD基因的人胰腺癌细胞毒性较低 ,在CD基因的原位转导的胰腺癌裸鼠移植瘤模型上亦可观察到抗肿瘤效应。结论 腺病毒介导CD基因 ,不仅转染效果强 ,而且可有效治疗胰腺癌。证实了酶解药物前体在实验性胰腺癌的治疗作用。