腺病毒载体介导四环素调控的DT/VEGF体系的基因治疗

Tetracycline controlled DT/VEGF system gene therapy mediated by adenovirus vector

目的研究重组腺病毒在四环素调控下传递 DT_(390)-VEGF_(165)及DT_(390)-VEGF_(exon7)融合基因治疗肝癌的可行性.方法构建4种四环素 tet-off 调控的 DT_(39)-VEGF_(165)或 DT_(390)-VEGF_(exon7)融合基因重组腺病毒载体,用脂质体介导包装出4种重组腺病毒,分别感染肝细胞癌 HepG2细胞,在没有四环素的情况下观察肝癌细胞的形态变化,用2种粗制重组腺病毒治疗荷肝癌裸鼠,用免疫组化鉴定融合基因的表达.结果在培养液中加入 3mg·L^(-1)的四环素可包装出携带毒素融合基因的重组腺病毒,用噬斑分析法测定重组腺病毒滴度均为1×10^(10)pfu·L^(-1);粗制重组腺病毒感染 HepG2细胞8h 后换以无四环素的正常培养液,发现72h 后 HepG2细胞发生病变,细胞死亡率达95%;感染细胞经白喉抗毒素-FITC 免疫荧光抗体染色均呈现黄绿色荧光;用粗制重组腺病毒AdCA13-tTA-TRE-DT_(390)-VEGF_(165)和 AdCA13-tTA-VEGF_(165)-DT_(390)-TRE 注射肝癌瘤体可观察到瘤体生长明显得到抑制,AFP 值显著下调,免疫组化结果显示融合基因在瘤体内得到了表达.结论重组腺病毒可以提高转基因效率.

AIM To study the feasibility of tetracycline-controlled recombinant adeno-virus transferring the fusion gene therapy on human liver cell cancers. METHODS Four replication-defective recombinant adenovirus vectors containing DT_(390)-VEGF_(165) or DT_(390)- VEGF_(exon7) gene under transcriptional control of a tetracycline-regulated system were constructed.Four recombinant adenovirus were packaged and used to infect HepG2 cells to observe the morphologic changes in the absence of tetracycline.Two recombinant adenovirus were injected into the established human liver neoplasms in NC nude mice respectively.The expression of chimeric gene in solid tumors was assayed by immunohist- ochemistry. RESULTS It was found that the recombinant adenovirus could be packaged by adding 3 mg·L^(-1) tetracycline into culture medium.Titer of each of the rough recombinant adenovirus was about 1×10^(10)pfu·L^(-1).8 h after infection by the rough recombinant adenovirus,we rinsed the infected HepG2 cells twice with PBS,and incubated them untill we found that virtually 95% HepG2 cells were killed in the activated state (i.e.without tetracycline) 72 h later.The infected cells showed yelow-green luminescence by immunofluorescence antibody dyeing with antidiph-therin-FITC.Tumor growth was inhibited and AFP was decreased in nude mice receiving injection of rough recombinant adenovirus AdCA13-tTA-TRE-DT_(390)- VEGF_(165) and AdCA13-tTA-VEGF_(165)-DT_(390)-TRE when compared to untreated control mice.Immunohistoch- emistry experiment showed tumor cells could be specifically stained by antidiphtherin-FITC,indicating that the chimeric gene can be expressed in the tumor. CONCLUSION The recombinant adenovirus could increase the gene transfer efficiency.