單一細胞層級腫瘤壞死因子與細胞膜上腫瘤壞死因子受體結合能力之研究

Single-Cell Study of Binding of Tumor Necrosis Factor α to TNF Receptor

李志明(Chi-Ming Lee)；石百祿(Pai-Lu Shih)；柯利(Mamadi-MS Colley)；洪仕淇(Shih-Chi Hung)；陳美燕(Mei-Yan Chen)；楊自森(Tzu-Sen Yang)；梁庭繼(Ting-Kai Leung)

轉錄因子 ； 腫瘤壞死因子 ； 腫瘤壞死因子受體 ； 單分子螢光技術 ； 藻紅素 ； Transcription factor ； Tumor necrosis factor ； Tumor necrosis factor receptor ； Single-molecule fluorescence technology ； Phycoerythrin

醫學與健康期刊

9卷1期（2020 / 03 / 01）

47 - 60

繁體中文

目的：Nuclear factor-κB（NF-κB）轉錄因子是一種控制DNA轉錄的蛋白複合體，包括p50、p52、p65（RelA）、RelB以及c-Rel。NF-κB可因不同的刺激而引起反應，包括紫外線照射、細胞激素、自由基、氧化低密度脂蛋白及細菌或病毒抗原等。近期研究顯示腫瘤壞死因子TNF-α與細胞膜上腫瘤壞死因子受體（TNF receptor，TNFR）結合後，會誘導NF-κB進行對下游基因的調控，並且發現細胞受到TNF-α刺激後，NF-κB會由細胞質輸運到細胞核，然後再由細胞核回到細胞質的振盪變化，並進行特定基因的表現。研究目標將透過單分子螢光技術，探討TNF-α與TNFR的結合模式。方法：本研究將提出結合單分子螢光技術之單一細胞層級方法學來分析TNF-α與TNFR的結合能力，利用TNF-α抗體將藻紅素有機染劑與TNF-α進行螢光標定，藉由標定TNF-α最佳化的螢光觀測方法，將可釐清TNF-α與TNFR在空間時間上的結合模式。結果：實驗結果發現藻紅素有機染劑可有效利用TNF-α抗體來標定TNF-α，同時透過藻紅素有機染劑的光漂白現象可以證實TNF-α與TNFR結合後具有形成單體、二聚體或是三聚體的可能性。結論：本研究所建立的單一細胞層級方法學將有助於探索TNF-α與TNFR結合後對NF-κB動力學之空間時間作用關係。

Objectives. Nuclear factor-κB (NF-κB) transcription factor is composed of homodimers or heterodimers. The NF-κB family has five subunits, including p50, p52, p65 (RelA), RelB, and c-Rel. NF-κB activity can be induced by a variety of stimuli, including ultraviolet irradiation, cytokines, free radicals, oxidized low density lipoprotein (LDL), and bacterial or viral antigens. Incorrect regulation of NF-κB has been linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development. A recent study reveals that binding of tumor necrosis factorα(TNF-α) to tumor necrosis factor receptor (TNFR) activates transcription of its target genes. In addition, after stimulation with TNF-α, NF-κB activation results in oscillations in the nucleolus and target gene expression can be regulated by negative feedback loops. The objective of this study was to monitor the binding mode of TNF-α to TNFR using a single-molecule fluorescence technique. Methods. In this investigation, we performed a single-cell study of binding of TNF-α to TNFR. We also highlighted our novel use of single-molecule fluorescence technology to study the spatiotemporal interaction in binding of TNF-α to its receptor at the single-cell level. To do this, we applied biotin anti-human TNF-α antibody to bind with TNF-α followed by chemical biding for Streptavidin Phycoerythrin (PE) to identify the structure of the space between TNF-α and TNFR. Results. Real-time imaging of single TNF-α molecule on the living NIH 3T3 cell surface revealed ligand-induced receptor dimerization and trimerization. Conclusion. This study introduces a new approach to probing the spatiotemporal interaction between bindings of TNF-α to TNFR and the corresponding NF-κB dynamics, potentially enhancing our understanding of TNF-α and TNFR interaction.

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