摘要
目的:利用血管内皮生长因子(VEGF)基因启动子介导鼠α1,3半乳糖基转移酶(α1,3GT)在缺氧肝癌细胞HepG2中靶向表达并检测其诱导的人血清天然抗体对肝癌细胞的杀伤效应,为肝癌的靶向基因治疗方法提供新的思路。方法:利用PCR从Balb/c小鼠肝组织中扩增出小鼠VEGF基因启动子片段并插入到荧光素酶报告载体中;用所构建的荧光素酶报告载体瞬时转染HepG2细胞;经缺氧诱导后检测转染细胞中荧光素酶活性;进一步通过基因重组构建受VEGF基因启动子调控的鼠α1,3GT重组逆转录病毒载体并稳定转染HepG2细胞;转染细胞在缺氧条件下培养后,通过CDCC试验检测人血清对转染细胞的杀伤效应。结果:在转染重组有VEGF基因启动子的荧光素酶报告载体的HepG2中,荧光素酶的活性是对照组的2-3倍;通过CDCC实验发现转染有鼠α1,3GT重组逆转录病毒载体的HepG2细胞经缺氧培养后对人血清的杀伤效应更加敏感。结论:利用VEGF基因启动子介导鼠α1,3GT在缺氧肿瘤细胞中靶向表达并诱导人血清天然抗体对肿瘤细胞产生杀伤效应在肿瘤的靶向基因治疗中具有潜在的临床应用价值。
Objective：To explore whether the murine alpha 1, 3 Galactosyltransferase gene （ α1,3 GT） expression driven by the VEGF promoter （pVEGF） was effective in inducing a killing effect of human serum to human liver cancer cell line HepG2 under the hypoxic condition. Methods ： pVEGF fragment was PCR - amplified from genome DNA of mouse liver and inserted into a luciferase reporter plasmid. The lueiferase reporter plasmid construct was then transfected into HepG2 cells. After exposure to hypoxia, luciferase activity in the transfected HepG2 cells was detected. Furthermore, a retroviral vector harboring the α1,3 GT driven by the pVEGF was constructed and transfected into HepG2 cells. The sensitivity of transfectant to human serum was tested by cytotoxicity assay under the hypoxic condition. Results ： A 2 - 3 - fold increase of luciferase activity was detected in HepG2 cells which were transfected with the luciferase reporter plasmid construct. HepG2 cells transfected with the retroviral vector which harbored the pVEGF and α1,3 GT gene were found more sensitive to human serum under the hypoxic condition. Conclusion：This study suggested a possible application of the murine α1, 3 GT gene driven by the pVEGF to the cancer gene therapy.
出处
《现代肿瘤医学》
CAS
2009年第4期598-601,共4页
Journal of Modern Oncology
基金
国家自然科学基金资助项目(30500604和30672391)
关键词
鼠α1
3半乳糖基转移酶
血管内皮生长因子
肝细胞癌
缺氧
murine alpha α1, 3 Galactosyhransferase gene
vascular endothelial growth factor
hepatocelluar carcinoma
hypoxia
