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首页> 外文期刊>World Journal of Gastroenterology >Construction of a regulable gene therapy vector targeting for hepatocellular carcinoma
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Construction of a regulable gene therapy vector targeting for hepatocellular carcinoma

机译：靶向肝细胞癌的可调控基因治疗载体的构建

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AIM: To construct a gene modified hepatocellular carcinoma (HCC) specific EGFP expression vector regulated by abbreviated cis-acting element of AFP gene. METHODS: The minimal essential DNA segments of AFP gene enhancer and promoter were synthesized through PCR from Genome DNA of HepG2 cells. Gene fragments were then cloned into the multiple cloning site of non-promoter EGFP vector pEGFP-1. Recombinant plasmid was transferred into positive or negative AFP cell lines by means of lipofectamine. The expression of EGFP was tested by fluorescence microscope and flow cytometry. The effect of all-trans retinoic acid (ATRA) on the expression of EGFP was tested in different concentrations. RESULTS: By the methods of restriction digestion and sequence analyses we confirmed that the length, position and orientation of inserted genes of cis-acting element of AFP were all correct. The transcription of EGFP was under the control of AFP cis-acting element. The expressing EGFP can only been detected in AFP producing hepatoma cells. The expression rate of EGFP in G418 screened cell line was 34.9+-4.1 %. 48 h after adding 1x10~(-7)M retinoic acid, EGFP expression rate was 14.7+-3.5 %. The activity of AFP gene promoter was significantly suppressed by addition of 1x10~(-7)M retinoic acid (P<0.05, P=0.003, t=6.488). CONCLUSION: This recombinant expression vector can be used as a gene therapy vector for HCC. The expression of tumor killing gene will be confined within the site of tumor and the activity of which can be regulated by retinoic acid.
机译：目的：构建由AFP基因的缩写顺式作用元件调控的基因修饰的肝细胞癌（HCC）特异性EGFP表达载体。方法：通过PCR从HepG2细胞基因组DNA中合成AFP基因增强子和启动子的最小必需DNA片段。然后将基因片段克隆到非启动子EGFP载体pEGFP-1的多克隆位点。借助脂质转染胺将重组质粒转移至阳性或阴性AFP细胞系中。用荧光显微镜和流式细胞仪检测EGFP的表达。在不同浓度下测试了全反式维甲酸（ATRA）对EGFP表达的影响。结果：通过限制性酶切和序列分析的方法，我们证实了AFP顺式作用元件插入基因的长度，位置和方向都是正确的。 EGFP的转录受AFP顺式作用元件的控制。只能在产生AFP的肝癌细胞中检测到表达的EGFP。在G418筛选的细胞系中，EGFP的表达率为34.9±4.1。加入1x10〜（-7）M视黄酸48小时后，EGFP表达率为14.7±3.5％。加入1x10〜（-7）M视黄酸可显着抑制AFP基因启动子的活性（P <0.05，P = 0.003，t = 6.488）。结论：该重组表达载体可作为肝癌的基因治疗载体。肿瘤杀伤基因的表达将被限制在肿瘤部位，其活性可以由视黄酸调节。

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