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背景与目的:与传统的单纯疱疹病毒胸苷激酶(herpessimplex virus thymidine kinase,HSV-TK)/丙氧鸟苷(ganci clovir,GCV)肿瘤自杀系统相比,人钠/碘同向转运体(human sodium-iodidesymporter,hNIS)是近年来发现的新型治疗基因。然而,两者的疗效尚需进一步提高。本研究旨在探索hNIS介导的放射性核素体系与HSV TK/GCV自杀基因体系对肝癌细胞的联合毒性作用。方法:构建EF1-α启动子调控下的hNIS的表达载体与CMV启动予调控下的GFP和HSV-TK共表达载体,慢病毒包装后转染肝癌细胞HepG2,荧光显微镜下观察重组肿瘤细胞HepG2/NTG荧光蛋白的表达;采用RT-PCR技术进一步检测目的基因hNIS和HSV-TK的表达情况;MTT检测GCV对HepG2/NTG的杀伤作用;摄碘试验及流出试验评价HepG2/NTG对碘的摄取及滞留情况;最后通过克隆形成实验评价GCV和~(131)I对HepG2/NTG的单独及联合杀伤作用。结果:RT-PCR技术、荧光显像证实hNIS、GFP和HSV-TK基因在肝癌细胞中成功表达;与对照组相比,HepG2/NTG的摄碘高出76倍,20 mi n摄碘率最高,其后表现出碘外流,有效半衰期不到10 min,同时这种碘摄取能被NaC1O_4特异性抑制。GCV或~(131)I对HepG2/NTG的毒性作用呈现剂量依赖性:50μg/mL GCV作用72 h后,实验组细胞的存活率仅为(33.98±2.71)%;放射性浓度为7.4 MBq/mL的~(131)I处理7 h后,细胞的存活率仅为(41.17±0.72)%;GCV和~(131)I联合作用后,细胞的存活率仅为(8.55±1.22)%,较单一药物作用细胞存活率下降了6倍。结论:~(131)I/GCV对重组肝癌细胞产生显著的毒性作用,提示慢病毒介导hNIS/TK基因共转染介导肿瘤的放射化学治疗是可行的。
BACKGROUND & AIM: Compared with the HSV-TK / GCV tumor suicide system, the human sodium / iodine symporter (human Sodium-iodidesymporter, hNIS) is a novel therapeutic gene discovered in recent years. However, the efficacy of both needs to be further improved. This study aimed to explore the combined toxicity of hNIS-mediated radionuclide system and HSV TK / GCV suicide gene system on hepatoma cells. Methods: The hNIS expression vector under the control of EF1-α promoter was co-expressed with the GFP and HSV-TK vectors under the control of CMV promoter. The lentiviral vector was packaged and transfected into HepG2 hepatocellular carcinoma cells. The expression of HepG2 / NTG fluorescent protein expression; RT-PCR technology to further detect the expression of the target gene hNIS and HSV-TK; MTT detection of GCV on HepG2 / NTG killing effect; iodine intake test and outflow test HepG2 / NTG evaluation of iodine uptake and Finally, the killing effect of GCV and ~ (131) I on HepG2 / NTG was evaluated by clonogenic assay. Results: The expression of hNIS, GFP and HSV-TK genes was confirmed by RT-PCR and fluorescence imaging in HepG2 cells. Compared with the control group, the uptake of HepG2 / NTG was 76 times higher than that of the control group , Followed by iodine efflux, the effective half-life of less than 10 min, while this iodine uptake can be NaC1O_4-specific inhibition. The toxicity of GCV or ~ (131) I to HepG2 / NTG in a dose-dependent manner: after 72 h of 50μg / mL GCV, the survival rate of the experimental group was only 33.98 ± 2.71%; the radioactive concentration was 7.4 MBq / mL The cell viability was only (41.17 ± 0.72)% after treated with ~ (131) I for 7 h. The survival rate was only (8.55 ± 1.22)% after GCV and ~ (131) Drug effect Cell viability decreased by 6 times. CONCLUSION: ~ (131) I / GCV has a significant cytotoxic effect on human hepatocellular carcinoma cells, suggesting that lentivirus-mediated cotransfection of hNIS / TK gene mediated tumor radiotherapy is feasible.