TNFRⅡ转基因诱导喉鳞状细胞癌裸鼠移植瘤模型肿瘤细胞凋亡的实验研究

目的：探讨肿瘤坏死因子受体2（tumor necrosis factor receptor 2，TNFRⅡ）转基因结合TNFα局部注射诱导喉鳞状细胞癌裸鼠模型肿瘤细胞的凋亡和杀伤肿瘤的效果，为喉鳞状细胞癌的基因治疗开辟新途径。方法：在利用人喉鳞状细胞癌Hep2细胞建立人喉鳞状细胞癌裸鼠移植瘤模型的基础上，以脂质体为载体采用体内转基因技术，活体转染人类TNFRⅡ并联合局部TNF-α注射，采用肿瘤大体观察、流式细胞术、免疫组织化学、TuneⅠ、透射电镜等方法观察TNFRⅡ转染前后肿瘤细胞TNFRⅡ表达水平、肿瘤细胞凋亡情况，综合评价对肿瘤杀伤和抑制的效果。结果：裸鼠喉癌移植瘤模型的成瘤率为94．5％。活体转基因48h TNFRⅡ表达水平达最高峰并在72h内维持较高水平。免疫组织化学方法证实TNFRⅡ主要表达在肿瘤细胞的细胞膜上，空质粒转染组肿瘤细胞几乎无TNFRⅡ表达。2000U TNFα局部注射对肿瘤的诱导凋亡和杀伤的抑制效率最高，表现为在TNFRlI转基因组动物治疗结束时肿瘤的体积为（1161．333±166．555）mm^3，瘤体重量为（1．100±0．832）g，瘤／鼠重为0．044±0．332，凋亡率为（38．226±13．671）％，与空质粒转染对照组相比具有明显差别。Tunel和超微结构观察发现前者的细胞凋亡明显高于后者。结论：通过TNFRⅡ活体转基因提高其蛋白表达水平可以明显提高TNFα局部注射杀伤喉癌移植瘤模型动物肿瘤细胞的效果，其机制是通过更有效诱导细胞凋亡来实现的。活体TNFRⅡ转基因结合TNFα局部注射有可能成为喉癌基因治疗的一个有效方法。

Induction of apoptosis by tumor necrosis factor receptors 2 transgene in human laryngeal squamous cacinoma in nude mice animal model

Objective：To investigate the effect of tumor necrosis factor receptors Ⅱ （TNFRⅡ） in vivo trans gene with topical injection of TNFα in inducing apoptosis and cell killing of laryngeal squamous carcinoma in nude mice animal model. Method： Laryngeal carcinoma impantation animal model was established on nude mice. In vivo gene transfection of TNFRⅡ was carried out using liposome as a carrier. TNFα was topically injected into tumor. Goss measurement of tumor, flowcytometry, immunohistochemistry, Tunel and transmission electron microscopy were conducted to observe the expression of TNFRⅡ protein and the apoptosis of tumor cells, and the effects of tumor killing and growth inhibition was objectively evaluated. Result：Nude mice models bearing laryngeal carcinoma was established in 94.5% animals. After in vivo gene transfection, the expression of TNFRⅡ protein reach the highest level at 48 hours, and remain in a substantially high level within 72 hours. Immunohistochemistry showed the expression of TNFRⅡ is mainly on the cell membrane of the transfected tumor cells. Topical injection of 2000 U TNFα was most efficient in inducing tumor cell apoptosis, cell inhibition and cell killing. The tumor volume, weight, and tumor/body ratio in TNFRⅡ transfected group were （1 161. 333±166. 555）mma , （1. 100± 0. 832）g and 0. 044± 0. 332, respectively, with a corresponding high level of tumor cell apoptosis rate （38. 226± 13. 671 ）%, all of which were significantly higher than that in non-transfected group. Tunel and ultrastructural ob servations demonstrated apoptosis related changes in the transfected tumor cells. Conclusion： Up-regulation of TNFRⅡ expression by in vivo gene transfection on tumor cells can remarkably enhance the tumor cell killing effect of topical injection of TNF-α In vivo transgene of TNFRⅡ in combination with topical injection of TNFα may become a effective gene therapy method in treating laryngeal cancer.