摘要
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、?
BACKGROUND:For most cases of laryngeal cancer,radical mutilating surgery and irradiation often yield poor results,especially for recurrent and metastatic tumors.Suicide gene therapy combined with immune gene therapy may offer possibilities to improve the patients' long term survival and quality of life. OBJECTIVE:To construct the expression vectors containing fusion genes from different combinations of the genes encoding herpes simplex virus thymidine kinase(HSV tk),recombinant human interleukin 2(rhIL 2),tumor necrosis factor(TNF α),which may provide a theoretical basis for developing gene therapies to reduce the recurrence,improve the curative effect and benefit the psycho social well being of the patients. DESIGN:A prospective experimental study was conducted. SETTING and PARTICIPANTS:The experiment on the fusion genes from different combinations of HSV tk,IL 2 and TNF αwas conducted in the Institute of Orthopedic Oncology of PLA,Tangdu Hospital,Fourth Military Medical University. INTERVENTIONS:The cDNA of human IL 2 was obtained by reverse transcriptional(RT) PCR.The three genes were amplified respectively by PCR and sequenced.By means of directional cloning the sequenced genes were subcloned into corresponding restriction sites of pLXSN in succession to generate different eukaryotic expression vectors. MAIN OUTCOME MEASURES:The amplification of each target gene,enzymatic and PCR identification of the target vectors. RESULTS:Double endonuclease digestion by EcoRI and BamH yielded an 2.07 kb fusion gene fragment from pL(TIT)SN,a 1.58 kb fragment from pL(TI)SN with the use of EcoRI and XhoI,a 1.59 kb fragment from pL(TT)SN with EcoRI and BamH,and a 1.13 kb fragment with pL(TK)SN with EcoRI and BamHI.By way of restriction digestion and PCR,the length,position and orientation of the inserted genes and fusion genes were all confirmed. CONCLUSION:Suicide gene combined with immune gene therapy is more beneficial for the patients with regard to their quality of life than irradiation and chemotherapy,and this study also provides a theoretical basis for gene therapy of tumor.
出处
《中国临床康复》
CSCD
2004年第14期2780-2781,F013,共3页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助项目(39970788)~~
