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[目的]构建TNFR1及其突变体重组质粒,研究TNFR1胞浆段结构域与sTNF-α肿瘤生物学功能的关系。[方法]通过RT-PCR和重叠PCR,构建TNFR1-pEGFP-N1,Y236A-TNFR1-pEGFP-N1、ΔNSD-TNFR1-pEGFP-N1和ΔDD-TNFR1-pEGFP-N1。采用荧光显微镜观察TNFR1内吞、流式细胞术检测表达率、MTT比色法检测胞毒。[结果]获得了TNFR1全长基因及相关突变体的重组质粒,无任何其他点突变,移码及缺失突变。以这些质粒为工具进行生物学检测,提示野生型TNFR1可以介导sTNF-α内化,Y236A-TNFR1却不能,ΔNSD-TNFR1和ΔDD-TNFR1同样影响sTNF-α对靶细胞的胞毒作用。[结论]TNFR1内化结构域、NSD和DD功能域对sTNF-α杀伤肿瘤细胞的功能起着重要的作用。该研究有助于进一步认识sTNF-α介导的杀瘤效应与TNFR1的关系,并为深入研究tmTNF-α通过TNFR1介导的杀瘤机制提供重要的实验工具,从而为肿瘤基因治疗提供新的靶点。
[Objective] To construct the recombinant plasmid of TNFR1 and its mutants and study the relationship between the cytoplasmic domain of TNFR1 and the biological function of sTNF-α tumor. [Methods] TNFR1-pEGFP-N1, Y236A-TNFR1-pEGFP-N1, ΔNSD-TNFR1-pEGFP-N1 and ΔDD-TNFR1-pEGFP-N1 were constructed by RT-PCR and overlap PCR. Endocytosis of TNFR1 was observed by fluorescence microscopy. The expression of TNFR1 was detected by flow cytometry. Cytotoxicity was detected by MTT assay. [Result] The full length of TNFR1 gene and related recombinant plasmids were obtained, without any other point mutation, frameshift and deletion mutation. Using these plasmids as a tool for bioassay, it was suggested that wild-type TNFR1 could mediate the internalization of sTNF-α, but Y236A-TNFR1 could not, ΔNSD-TNFR1 and ΔDD-TNFR1 also affected cytotoxicity of sTNF-α on target cells. [Conclusion] The internalization of TNFR1 domain, NSD and DD domains play an important role in the function of sTNF-α killing tumor cells. This study will help to further understand the relationship between TNF-α and TNF-α mediated by sTNF-α, and provide a new experimental tool for further study of tmTNF-α through tumor necrosis factor (TNFR1) -mediated tumorigenesis. Targets.