双靶向组织特异性HSV-tk/GCV抗瘤系统的构建及体外效应研究

目的：构建高靶向性基因转移及表达系统，并研究其介导HSVtk/GCV自杀基因系统对肝癌细胞的体外杀伤作用。方法： 通过重组技术分别构建antiTfR ScFvGAL4融合蛋白表达载体ScFvGAL4pET28a及含AFP启动子、GAL4特异性识别序列（GAL4rec）的HSVtk真核表达载体pEBAF/tkGAL4rec。IPTG诱导后，利用受体介导内吞作用介导pEBAF/tkGAL4rec质粒转染表面均高表达TfR的人肿瘤细胞株HepG2，SMMC7721及A549。潮霉素筛选后，MTT法检测GCV对它们的杀伤作用。结果：双酶切鉴定、SDSPAGE电泳及测序分别证明antiTfR ScFvGAL4融合蛋白及重组pEBAF/tkGAL4rec真核表达质粒构建成功。体外杀伤实验中，高分泌AFP(845 ng/ml) 的HepG2/tk细胞对GCV很敏感，且抑制率与GCV浓度及作用时间呈正相关；而低分泌AFP(2 ng/ml)的SMMC7721/tk细胞对GCV低度敏感；不分泌AFP 的A549/tk细胞则不敏感。结论：双靶向组织特异性HSVtk/GCV抗瘤系统构建成功，并显示出很好的靶向性。

The Construction and Biological Effects of Double Targeting Tissue-Specific HSV-tk/GCV Anti-Tumor System in vitro

Objective: To construct the potent targeting gene delivery and expression system, and to investigate the special killing effect of HSV-tk/GCV system on human liver cancer cells in vitro mediated by it. Methods:The anti-TfR ScFv-GAL4 fusion protein expression vector ScFv-GAL4-pET28a and the eukaryocyte expression plasmid pEBAF/tk-GAL4rec were constructed by recombinant DNA technology. After the induction by IPTG, we got the anti-TfR ScFv-GAL4 fusion protein as delivery vector to transfect pEBAF/tk-GALArec into the human liver cancer cells line HepG2,SMMC7721 and lung cancer cells line A549 which all over-express TfR via receptor-mediated endocytosis. The positive cell clones were selected by hygromycin and were named HepG2/tk,SMMC7721/tk and A549/tk respectively. Then MTT method was used to determine the killing effect of GCV on them. Results: The constructions of the ScFv-Gal4 fusion protein and the recombination expression plasmid pEBAF/tk-GAL4rec were confirmed by double enzyme digestion, SDS-PAGE and sequencing. In the experiment of cytotoxicity effect, the HepG2/tk cells that over-secrete AFP ( 845 ng/ml) were highly sensitive to the toxicity of GCV, whereas the SMMC7721/tk cells that under-secrete AFP (2 ng/ml) were slightly sensitive to GCV, and the A549/tk cells that don t secrete AFP were not. Conclusions:The double-targeting and tissue-specific HSV-tk/GCV anti-tumor system has been constructed successfully, and it displays a good targeting to tumor cells.