基因-病毒治疗系统CNHK300-murine endostatin的构建及体外研究

Development of gene-viral therapeutic system CNHK300-mE and its primary study in vitro

目的构建一种新型的肿瘤基因-病毒治疗系统CNHK300-murine endostatin(CNHK300-mE),以肿瘤增殖病毒为载体,携带抗肿瘤新生血管生成的基因。方法利用基因重组技术将人端粒酶逆转录酶(hTERT)启动子插入腺病毒E1A基因上游,将小鼠内皮抑素基因表达盒插入到腺病毒包装信号和hTERT启动子之间,通过病毒增殖试验、电镜技术、Western blot分析、酶联免疫吸附实验(ELISA)、鸡胚绒毛尿囊膜(CAM)试验,观察CNHK300-mE的复制情况、mE的表达情况及对鸡胚绒毛尿囊膜新生血管的抑制情况。结果成功构建了基因-病毒治疗系统CNHK300-mE,该系统为hTERT启动子调控腺病毒E1A基因表达并携带小鼠内皮抑素基因的重组腺病毒。该病毒可以在端粒酶阳性的胃癌细胞株SGC-7901和肝癌细胞株HepGII中大量增殖及复制,而在端粒酶阴性的正常纤维细胞中不增殖。电镜证实该重组病毒在胃癌细胞株SGC-7901中复制及增殖。ELISA检测表明SGC-7901感染CNHK300-mE后7d,小鼠内皮抑素的表达量为1000μg/L。CAM实验证实该病毒感染胃癌细胞后的培养液上清具有抗新生血管生成的生物活性。结论基因-病毒治疗系统CNHK300-mE能在端粒酶阳性的肿瘤细胞中特异性增殖复制,并大量表达具有抗新生血管生成生物活性的小鼠内皮抑素。

Objective To develop a novel gene-viral therapeutic system CNHK300-murine endostatin （CHNK300-mE） and to investigate its selective replication in telomerase-positive cancer cells and production of mouse endostatin. Methods A novel gene-viral therapeutic system named CHNK300-mE was constructed by employing human telomerase reserve transcriptase （hTERT） promoter to drive the expression of adenovirus E1A gene and cloning the murine endostatin gene into the adenovirus genome. The expression of the antiangiogenic gene of murine endostatin and the suppression of angiogenesis were observed respectivedly by virus replication assay in vitro, electron microscopy, Western blot, and chicken embryo chorioallantoic membrane （CAM） assay. Results A new kind of gene-viral vector system, designated CNHK300-mE was constructed. The novel vector system possessed the same property as the replicative virus ONYX-015, replication and proliferation in telomerase-positive cancer cells but not in the normal cells. When carrying the gene of mE, it made the expression of this gene in tumor cells far more effectively than the adenovirus vector employed in the traditional gene therapy. Electron microcopy con- firmed that this vector system replicated and proliferated in the gastric cancer cells. CAM assay demonstrated that the mE produced by CNHK300-mE infected gastric cancer cells possed antiangiogenic bioactivity. Conclusion CNHK300-mE, a novel vector in which the anntiangiogenic gene is inserted into the genome of the replicative virus specific for the tumor cells, can increase the expression level of antiangiogenic gene. It possesses the advantages of gene therapy and antiangiogenic therapy, thus further enhancing the curative effect and overcoming such disadvantages as low transfer rate, low expression, lack of target tropism.