癌胚抗原启动子调控融合自杀基因yCDglyTK载体的构建及其应用研究

Construction of carcino-embryonic antigen promotor controls the novel fusion suicide gene yCDglyTK and its application

目的构建靶向性基因治疗载体pcDNA3.1(-)CVyCDglyTK,研究癌胚抗原(CEA)启动子是否能控制新型融合自杀基因yCDglyTK在CEA阳性结肠癌细胞中专一性表达和杀伤作用。方法采用PCR、RT-PCR、融合PCR、酶切、连接等技术构建由CEA启动子、CMV增强子驱动的融合自杀基因pcDNA3.1(-)CVyCDglyTK表达载体和分别由CEA启动子和巨细胞病毒(CMV)增强子驱动的融合自杀基因pcDNA3.1(-)CEAyCDglyTK、pcDNA3.1(-)CMVyCDglyTK表达载体,以磷酸钙纳米为载体分别转染CEA阳性的人结肠癌细胞株LOVO细胞和CEA阴性的Hela细胞,采用RT-PCR、免疫荧光法检测感染细胞中yCDglyTK基因的表达,并用MIT法检测感染后细胞对5-氟胞嘧啶(5-FC)的敏感性。结果LOVO细胞在感染以上三种质粒表达载体后均有yCDglyTKmRNA表达,且对5-FC的敏感性明显增强,Hela细胞在纳米-pcDNA3.1(-)CMVyCDglyTK复合物感染后有yCDglyTKmRNA表达,对5-FC的敏感性增强,而在纳米-pcDNA3.1(-)CEAyCDglyTK及纳米-pcDNA3.1(-)CVyCDglyTK复合物感染后则没有yCDglyTKmRNA表达,5-FC对其亦无杀伤作用。结论该实验构建的由CEA启动子、CMV增强子驱动的融合自杀基因yCDglyTK靶向性基因治疗载体,能使融合自杀基因在CEA阳性细胞中专一性表达,从而达到靶向治疗肿瘤的目的。

[Objective] To construct targeted plasmid vector pcDNA3.1 （-）CV yCDglyTK and study whether carcino-Embryonic Antigen promotor determines the specific expression of the novel fusion suicide gene and specific lethal effect in CEA-producing colorectal carcinoma cells. [Methods] We constructed the expression plasmid vector of peDNA3.1 （-）CVyCDglyTK controlled under CEA promoter and cytomegalovirus（CMV） enhancer;, pcDNA3.1 （-） CEAyCDglyTK controlled under CEA promoter and pcDNA3.1 （-）CMVyCDglyTK controlled under CMV enhancer, with PCR, PT-PCR, confusion PCR, enzyme restriction, ligation and so on. We combined the calcium phosphate nanoparticles（CPNP） with suicide gene yCDglyTK for transfection the CEA-Positive cells （LOVO） and CEA-negative cells（Hela） respectively; The expression of the yCDglyTK gene was detected by RT-PCR and immunofluoreseenee Assay, MTT analysis was used to detect the eytotoxie effects of the yCDglyTK/5-FC system. [Results] The CEAproducing cells （human eoloreetal eareionma line LOVO cell） showed yCDglyTK mRNA expression and were sensitive to 5-Fe after infection with CPNP- peDNA·CV·yCDglyTK and CPNP-peDNA·CEA·yCDglyTK, CPNP-peDNA·CMV·yCDglyTK, respectively;However,the CEA-negative eells（Hela cell） had no effect and yCDglyTK mRNA expression. After infection with CPNP-peDNA-CV.yCDglyTK,CPNP-peDNA-CEA-yCDglyTK.but expressed yCDglyTK mRNA and sensitive to 5-FC after infection with CPNP- pcDNA·CMV·yCDglyTK. [Conclusion] The targted plasmid vector of pcDNA3. 1（-）CVCDglyTK controlled under CEA promoter and CMV enhancer could control the expression of fusion suicide gene in CEA-positive colorectal careionma cell, to achieve for target cancer gene therapy.