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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48hTNFRⅡ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。2000UTNFα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TNFRⅡ转基因组动物治疗结束时肿瘤的体积为(1161.333±166.555)mm3,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。
Objective: To investigate the effect of tumor necrosis factor receptor 2 (TNFRⅡ) transfection combined with local injection of TNFα on apoptosis and tumorigenesis of laryngeal squamous cell carcinoma in nude mice model. Gene therapy opens up new avenues. METHODS: Human laryngeal squamous cell carcinoma xenografts in nude mice were established by using human laryngeal squamous cell carcinoma Hep2 cells. The transfection of human TNFRⅡ and local TNF-α The expression of TNFRⅡ in tumor cells before and after TNFRⅡ transfection and the apoptosis of tumor cells were observed by tumor gross observation, flow cytometry, immunohistochemistry, Tune Ⅰ and transmission electron microscopy. The effects on tumor killing and inhibition were comprehensively evaluated. Results: The nude mouse laryngeal xenograft tumor model was 94.5%. The expression level of 48hTNFRII gene in living transgene reached its peak and maintained at a high level within 72h. Immunohistochemistry confirmed that TNFR Ⅱ was mainly expressed on the cell membrane of tumor cells. There was almost no TNFR Ⅱ expression in tumor cells transfected with empty plasmid. Topical injection of 2000UTNFα had the highest inhibitory effect on the induction of apoptosis and killing of tumor cells. The tumor volume was (1161.333 ± 166.555) mm3 and the tumor weight was (1.100 ± 0.832) g at the end of the TNFRII transgenic animal treatment. The tumor / The rat weight was 0.044 ± 0.332 and the apoptosis rate was (38.226 ± 13.671)%, which was significantly different from the control group. Tunel and ultrastructure observation found that the former apoptosis was significantly higher than the latter. CONCLUSION: Increasing the protein level by TNFRII gene transfection can significantly enhance the effect of local injection of TNFα on the killing of tumor cells in the model of laryngeal cancer. The mechanism is through more effective induction of apoptosis. Local injection of TNFRII transgene combined with TNFα may be an effective method for gene therapy of laryngeal cancer.