Jin Sisi, Lin Xianfan, Guan Huaqin, Wu Jinming
Department of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang 325000, P.R. China.
Exp Ther Med. 2015 Mar;9(3):863-867. doi: 10.3892/etm.2015.2166. Epub 2015 Jan 2.
The aim of the present study was to construct a gene-modified hepatocellular carcinoma (HCC)-specific analgesic-antitumor peptide (AGAP) expression vector regulated by the α-fetoprotein (AFP) promoter and enhancer, in order to evaluate its effect. The AFP promoter is generally used in HCC-specific gene therapy strategies. However, this approach is limited by the weak activity of the AFP promoter. Linking the AFP enhancer and promoter has been shown to generate a stronger and more HCC-selective promoter. The AGAP DNA fragment was amplified from the total RNA of the Chinese scorpion, Karsch. The fragment was subsequently cloned into the pAFP plasmid with the minimal essential DNA fragment, which included the AFP gene promoter and enhancer, to construct the recombinant plasmid, pAFP-AGAP. The plasmid was transfected into HepG2 cells and the mRNA expression levels of AGAP were detected by reverse transcription polymerase chain reaction (RT-PCR). In addition, Cell Counting Kit 8 (CCK-8) was used to analyze the cytotoxicity of plasmid transfection. The length, position and orientation of the inserted AGAP gene were all confirmed to be correct; thus, the recombinant vector was successfully constructed. Using RT-PCR and CCK-8 analysis, the mRNA expression levels of AGAP and the cytotoxicity in AFP-producing human HCC cells were determined. The AFP promoter and enhancer were found to specifically accelerate the expression of the target genes within the cells that were positive for AFP. Therefore, the method used in the present study was demonstrated to be a novel integration of traditional Chinese medicine with western medicine.
本研究的目的是构建一种由甲胎蛋白（AFP）启动子和增强子调控的基因修饰的肝细胞癌（HCC）特异性镇痛抗肿瘤肽（AGAP）表达载体，以评估其效果。AFP启动子通常用于HCC特异性基因治疗策略。然而，这种方法受到AFP启动子活性较弱的限制。已证明连接AFP增强子和启动子可产生更强且更具HCC选择性的启动子。AGAP DNA片段从东亚钳蝎的总RNA中扩增得到。随后将该片段克隆到含有最小必需DNA片段（包括AFP基因启动子和增强子）的pAFP质粒中，构建重组质粒pAFP-AGAP。将该质粒转染到HepG2细胞中，通过逆转录聚合酶链反应（RT-PCR）检测AGAP的mRNA表达水平。此外，使用细胞计数试剂盒8（CCK-8）分析质粒转染的细胞毒性。插入的AGAP基因的长度、位置和方向均被证实正确；因此，成功构建了重组载体。通过RT-PCR和CCK-8分析，测定了AGAP在产生AFP的人HCC细胞中的mRNA表达水平和细胞毒性。发现AFP启动子和增强子可特异性加速AFP阳性细胞内靶基因的表达。因此，本研究中使用的方法被证明是中西医结合的一种新方法。
