舌癌细胞系T-TFL的建立和生物学性状检测

背景基因治疗已成为恶性肿瘤研究领域的热点和发展趋势,但舌癌基因治疗的研究报道极少.目的构建含相关死亡结构域蛋白(FADD)及肿瘤坏死因子受体1(TNFR1)基因功能结构域的融合基因TFL,稳定转染入人舌鳞状细胞癌细胞系(Tca-8113)中,检测建系细胞T-TFL的生物学性状,探讨一种更有利于舌癌患者治疗期及治疗后期内生活质量的治疗手段.设计以诊断为依据,前瞻性研究.地点和对象实验在第四军医大学口腔医学院口腔颌面外科完成,研究对象为人舌鳞状细胞癌细胞系(Tca-8113),上海第二医科大学口腔医学院建系.干预反转录PCR获得人FADD及TNFR1基因cDNA,重组PCR法构建含二者功能结构域的融合基因TFL,通过阳离子脂质体法稳定转染TFL基因入Tca-8113细胞中.主要观察指标Westernblot检测融合蛋白TNFR1/DED表达,通过形态学观察、生长曲线等检测T-TFL细胞的生物学性状.结果获得了人FADD及TNFR1基因并构建成功融合基因TFL,转染入Tca-8113细胞后,能表达融合蛋白TNFR1/DED活性,且T-TFL细胞与亲本Tca-8113细胞的生物学性状无明显差异.结论T-TFL细胞能表达融合蛋白TNFR1/DED活性,可以为进一步深入研究舌癌基因治疗提供实验基础.

Identification of construction and biological characters of tongue carcinoma cell line T-TFL

BACKGROUND: The application of gene therapy has brought new hopes to the cure of cancer, but there were still few reports on tongue carcinoma gene therapy.OBJECTIVE: To construct the fusion gene TFL including the partial domains of Fas-associated death domain protein(FADD) and tumor necrosis factor receptor 1 (TNFR1) gene, then transfected it into human tongue squamous carcinoma cell line(Tca-8113) to construct and identify the cell line T-TFL which contained the fusion gene TFL in order to establish a means of improving the tongue carcinoma patients. To explore a therapeutic means in favour of the tongue carcinoma patient' s survival quality.DESIGN: A prospective study was performed according to the diagnosis.SETTING and PARTICIPANTS: The experiment was completed in Department of Oral and Maxillofacial Surgery, College of Stomatology, Fourth Military Medical University. The subject was human tongue squamous carcinoma cell line Tca-8113 established by College of Stomatology, Shanghai Second Medical Uniersity.INTERVENTIONS: RT-PCR was used to obtain cDNA of human FADD and TNFR1 gene. and recombinant PCR was used to construct TFL fusion gene;then transfected the fusion gene into Tca-8113 cells stabilized by plasmid-liposome complex transduction method.MAIN OUTCOME MEASURES: The fusion protein TNFR1/DED expression was detected by western blot; the morphological obervation and growth curve assay were used to evaluate the biological characters of T-TFL cell.RESULTS: Human FADD and TNFR1 gene were obtained respectively, the fusion gene TFL was constructed successfully. The T-TFL cell could expressed the fusion protein TNFR1/DED after TFL gene was transfected into it, and there were no obvious differences in biological characters between T-TFL and Tca-8113 cells.CONCLUSION: The construction of the fusion gene TFL and the T-TFL cell line could express the fusion protein TNFR1/DED, which could afford a novel experimental basis for human tongue carcinoma gene therapy.