摘要
目的 :构建含FADD及TNFR1基因功能结构域的融合基因TFL ,稳定转染入舌癌细胞后 ,通过体内、外实验初步观察融合蛋白TNFR1/DED在重组人肿瘤坏死因子 α(rhTNF α)作用下介导舌癌细胞凋亡的效应。方法 :反转录及重组PCR构建融合基因TFL ;鉴定融合蛋白TNFR1/DED在建系舌癌细胞T TFL中的表达 ,通过MTT法、形态学观察、DAN片段及体内抑瘤实验 ,观察融合蛋白TNFR1/DED介导舌癌细胞凋亡的效应。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 ,体外实验观察到rhTNF α可有效地杀伤舌癌细胞 ,体内可明显抑制移植瘤的生长 ,且荷瘤动物内脏器官无病理性改变。结论 :融合蛋白TNFR1/DED可有效地介导舌癌细胞凋亡 ,为舌癌基因治疗研究提供实验基础。
Objective:To study of TNFR1/DED fusion protein mediated a pp optosis in tongue carcinoma Tca 8113 cells. Methods:RT-PCR and recombinant PCR were used to construct TFL fusion gene,the reconstructed gen e was transfected into Tca 8113 cells then the fusion protein TNFR1/DED expressi on in the cells was detected by Western blot. The cells were treated with recomb inant human tumor necrosis factor-α(rhTNF-α) at 0.01-1 000 ng/ml for 48 h,M TT assay, electron microscopy, flow cytometry, DNA fragmentation assay and in vivo antitumor activity assay in nude mice were used to evaluated the effects of TNFR1/DED fusion protein mediated appoptosis in the cells. Results:Human FADD and TNFR1 gene were obtained, fusion gene TFL was c onstructed.Fusion protein TNFR1/DED was expressed in Tca 8113 cells after TFL gene had been transfected into the cells.In the in vitro and in vivo experiments,rhTNF-α showed much stronger cytotoxic and antitumor effects and i nduced much higher appoptosis ratio on TFL gene transfected cells(T-TFL) th an on the blank vector transfected cells(T-pc) and the parental cells(Tca 8113) .No pathological change was observed in lung,liver,heart and kidney in all the n ude mice.Conclusion:The TNFR1/DED fusion protein may mediate app optosis of tongue carcinoma cells induced by rhTNF-α.
出处
《实用口腔医学杂志》
CAS
CSCD
北大核心
2005年第1期62-66,共5页
Journal of Practical Stomatology
