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核糖体基因区打靶载体pHrn是本室构建的人源基因载体之一,为探讨其在肝癌基因治疗的作用,本研究构建了pHr-CeTpCDUPRT/GFP质粒载体并进行了肝癌的体内外治疗实验研究.该质粒载体以pHrn载体为骨架,CMV+hTERT启动子作为转录调控元件,CDUPRT/GFP自杀融合基因为目的基因.体外转染肝癌细胞BEL7402后,流式细胞仪检测转染效率为30%~50%;RT-PCR和Westernblotting结果表明,有CDUPRT表达;HPLC检测5-FC可转化为5-FU,给药后12h5-FU浓度达60.15μg/mL;四唑盐(Methylthiazolyltetrazolium,MTT)分析结果显示,200~800μg/mL5-FC使BEL7402的平均存活率为60%~35%.同时,对裸鼠肝癌模型进行瘤内转染,结果显示,当持续注射质粒和前体药物治疗时,裸鼠肿瘤生长明显被抑制,甚至体积稍有缩小;RT-PCR检测结果表明,瘤内有CDUPRT表达;HPLC检测裸鼠血清中的5-FU浓度为7.694μg/mL;肿瘤组织病理切片显示,大量肿瘤细胞坏死.这些结果为实际应用此载体进行肝癌基因治疗提供了重要的实验依据.
In order to explore its role in gene therapy of hepatocellular carcinoma, we constructed pHr-CeTpCDUPRT / GFP plasmid vector and carried out in vivo and in vitro treatment of hepatocellular carcinoma The plasmid vector was constructed with pHrn vector as backbone, CMV + hTERT promoter as transcriptional regulatory element and CDUPRT / GFP suicide fusion gene as target gene.Transfection of BEL7402 hepatocellular carcinoma cells in vitro showed that the transfection efficiency was 30% 50%. The results of RT-PCR and Westernblotting showed that CDUPRT was expressed. The 5-FC could be converted into 5-FU by HPLC, and the concentration of 5-FU was 60.15μg / mL at 12h after administration. The results of MTT The results showed that the average viability of BEL7402 was 200% ~ 800μg / mL for 60% -35% of the BEL7402 cells.At the same time, the transfection of the hepatocellular carcinoma model in nude mice was performed in vivo and the results showed that when the plasmid and prodrug were continuously injected The tumor growth was significantly inhibited and even the volume was slightly reduced. The results of RT-PCR showed that there was CDUPRT in the tumor, the concentration of 5-FU in the serum was 7.694 μg / mL by HPLC. The pathological sections of tumor showed that a large amount of Tumor cell necrosis. These results are realistic Application of this vector for liver cancer gene therapy provides an important experimental basis.