【摘要】 目的:分别构建甲胎蛋白(α- fetoprotein,AFP)启动子AF 0 3和AFP杂合启动子[HRE]AF调控的 PNP基因表达载体,检测并分析两者的表达差异性。方法: 将AF 0 3和[HRE]AF启动子插入载体 pcDNA- 3 .0 中,构建肝癌特异表达载体pAF- 0. 3 和 p[HRE]AF;将 PNP基因分别插入 pAF -0 .3 和 p[HRE] AF中,构建两启动子调控的 PNP基因表达载体;通过酶切、PCR及测序鉴定各重组体。用脂质体介导法将两重组体转染不同细胞株,RT PCR检测 PNP基因在各细胞株中表达,分析两者表达的特点。结果: 两目的片段均正确插入相应载体,pAF- 0 3/PNP中的 PNP基因在 AFP阳性肝癌细胞中实现了靶向性表达,而p[HRE] AF/PNP 中的 PNP 基因则在AFP阳性和阴性肝癌细胞中实现了靶向性表达。结论: 两种 PNP基因表达载体是肝癌基因治疗中新型、高效、特异性的治疗载体。两者的成功构建为利用PNP/Mep -dR系统进行肝癌的靶向性基因治疗奠定了坚实的基础。更多还原

【Abstract】 OBJECTIVE:To construct two expression vectors harboring PNP gene under a α-fetoprotein (AFP)tissue-specific promoter AF 0.3 and a hybrid AFP promoter [HRE]AF,and to detect and analyze their expressions in different cell lines. METHODS: AF 0.3 and [HRE]AF promoter were inserted into pcDNA 3.0 vector, and two recombinant vectors controlled by two different AFP promoters, pAF 0.3 and p[HRE]AF, were constructed. PNP gene were inserted into pAF 0.3 and p[HRE]AF vectors separately, two PNP gene expression vectors driven by two different promoters, pAF0.3/PNP and p[HRE] AF/PNP, were constructed by using recombinant DNA techniques. The recombinants were analyzed and identified by restriction enzyme, PCR and sequencing. Two PNP gene expression vectors were transfected into four different cell lines by the liposome-mediated method. The expressions of PNP gene in all cell lines were determined by RT-PCR method. RESULTS: All target fragments were separately cloned into corresponding vectors. The target PNP gene expression of pAF 0.3/PNP in AFP positive hepatocellular carcinoma cell line was detected. As for p[HRE]AF/PNP, the target PNP gene expressions were detected both in AFP positive and negative hepatocellular carcinoma cell lines.CONCLUSIONS:The two PNP gene expression vectors driven by AFP promoters are novel, effective and specific vectors for human hepatocellular carcinoma gene therapy. The successful constructions of two vectors will provide a substantial basis for the target gene therapy of hepatocellular carcinoma with the PNP/Mep-dR system and the AFP promoter.更多还原