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摘要背景:目前喉癌的治疗方法对于晚期复发和远隔转移病例难以获得满意效果,自杀基因联合免疫基因疗法可能能够长期提高肿瘤患者的生存率及生活质量。目的:构建含有单纯疱疹病毒胸苷激酶基因(HSV-tk)、重组人白介素2(rhIL-2)、肿瘤坏死因子-α(TNF-α)(rhTNF-α)不同组合融合基因的反转录病毒表达载体,寻找一个更有利于患者生存期内不易复发、根治效果良好、有利于社会、心理、生活质量基因治疗的理论基础。设计:前瞻性实验研究。地点和对象:实验在第四军医大学唐都医院全军骨肿瘤研究所完成,对象为HSV-tk、白介素2(IL-2)、TNF-α融合基因。干预:以RT-PCR制备人IL-2cDNA,分别以PCR方法扩增3目的基因,扩增产物纯化、测序后以定向克隆方法依次将各片段正向插入至pLXSN表达载体的多克隆位点间,以构建含不同融合基因的真核表达载体。主要观察指标:各目的基因PCR扩增结果,目的载体的酶切鉴定,PCR鉴定目的载体。结果:pL(TIT)SN经EcoRI,BamHI双酶切得到大小约2.07kb融合基因片段,pL(TI)SN经EcoRI,XhoI酶切得到大小约1.58kb融合基因片段,pL(TT)SN经EcoRI,BamHI双酶切得到大小约1.59kb融合基因片段,pL(TK)SN经EcoRI,BamHI双酶切得到大小约1.13kb片段。经限制性酶切分析及PCR方法鉴定,各插入基因及融合基因大小、?
Abstract BACKGROUND: At present, the treatment of laryngeal cancer is difficult to obtain satisfactory results for patients with late recurrence and distant metastasis. Suicide gene combined with immune gene therapy may improve the survival rate and quality of life of cancer patients in the long term. OBJECTIVE: To construct a retroviral vector containing different combinations of HSV-tk, rhIL-2, and TNF-α (rhTNF-α) Virus expression vector, looking for a more conducive to patients with recurrence is not easy to survive, cure well, is conducive to social, psychological, quality of life gene therapy theoretical basis. Design: Prospective Experimental Study. Location and Subjects: The experiment was performed at the National Institute of Bone Tumor of the Tangdu Hospital of the Fourth Military Medical University for the HSV-tk, interleukin 2 (IL-2) and TNF-α fusion genes. Intervention: Human IL-2 cDNA was prepared by RT-PCR, and 3 genes were amplified by PCR respectively. The amplified products were purified and sequenced. The fragments were inserted into the multiple cloning sites of pLXSN expression vector in turn by directional cloning method To construct eukaryotic expression vectors containing different fusion genes. MAIN OUTCOME MEASURES: PCR amplification results of each gene of interest, restriction enzyme digestion of the target vector and identification of the target vector by PCR. Results: The pL (TIT) SN was digested with EcoRI and BamHI to get the 2.07 kb fusion gene fragment. The pL (TI) SN fragment was digested with EcoRI and XhoI to obtain 1.58 kb fusion gene fragment. The pL (TT) , BamHI double digestion to obtain a size of about 1.59kb fusion gene fragment, pL (TK) SN by EcoRI, BamHI double digestion to obtain a fragment of about 1.13kb in size. Restriction enzyme digestion and PCR were used to identify the size of each inserted gene and fusion gene.