免疫系統是一個保護身體免於感染的複雜網絡，免疫反應失調可能會造成自體免疫疾病、免疫缺陷及癌症，因此，能夠調節免疫反應的工具是目前非常需要的。為了能夠非侵入地調控特定區域的免疫反應，我們欲建立一套超音波調控免疫反應的系統。我們的團隊最近開發出一種聲波遺傳技術，透過置換聽覺感知蛋白Prestin的兩個胺基酸序列(N7T和N308S)後可以使被轉染的細胞具有超音波感應能力，並引發鈣離子流入細胞內。為此，我們同時結合此技術和鈣離子敏感的NFAT啟動子，並建構了三種依賴NFAT啟動子表現基因的質體，分別具有活化巨噬細胞、活化胞殺T細胞及清除癌細胞的功能，期望透過超音波刺激活化NFAT訊號途徑來促進NFAT下游的特定基因表達。我們測試此系統在RAW264.7細胞、cbMSC細胞及293T細胞的可行性，結果顯示只有在被轉染的293T細胞中可以藉由兩小時的超音波刺激促進NFAT下游的基因表現。未來我們將透過修飾NFAT啟動子來提升NFAT途徑的敏感度及下游基因的表現。

The immune system is a complicated network protecting the body against infection. Dysfunction of the immune responses leads to autoimmune diseases, immunodeficiencies, and cancers. Therefore, the tools to modulate the immune responses are highly desired. In this study, we aimed to develop a new tool to regulate the immune response noninvasively and spatiotemporally. Recently, our group has established a sonogenetic system that used the focused ultrasound to trigger the calcium response of genetically defined cells. This is achieved by an engineered auditory-sensing protein, Prestin with two amino acid substitutions (N7T, N308S) (Huang et al., 2020). Taken advantage of this, we introduced the engineered Prestin and immune mediators that can be triggered by a calcium-sensitive NFAT promoter for sonogenetically controlling gene of interest expression. We have constructed three different plasmids designed for macrophage activation, T cell activation, and cancer cell elimination, respectively, then tested the feasibility of our system in RAW264.7 cells, cbMSCs, and 293T cells. However, only transiently transfected 293T cells can be induced at about 2.5-fold expression compared with the control samples after 2-hour ultrasonic stimulation. The improvement of NFAT-driven gene induction by modified NFAT promoter will be studied in the future.

摘要 i
Abstract ii
致謝 iii
Table of Contents iv
Chapter 1 Introduction 1
1.1 Overview of the immune system 1
1.2 Summary of conventional approaches used to manipulate immune responses 3
1.3 Mesenchymal stem cells and immune modulation 6
1.4 Tumor Necrosis Factor-Related Apoptosis Ligand (TRAIL) and cancer treatment 7
1.5 Calcium and Nuclear factor of activated T-cells (NFAT)-signaling pathway 8
Chapter 2 Material and methods 11
2.1 Cell culture and transfection 11
2.2 Generation of DNA constructs 12
2.3 Lentivirus preparation 13
2.4 Reverse transcription quantitative real-time PCR (qPCR) 14
2.5 Enzyme-linked immunosorbent assay (EILSA) 15
2.6 Ultrasound stimulation 15
2.7 Statistical analysis 16
Chapter 3 Results 17
3.1 The design of our sonogenetic system 17
3.2 Calcium ionophore serves as a positive control to trigger calcium influx. 19
3.3 Ultrasound treatment is insufficient to activate NFAT signaling pathway. 23
3.4 Using mesenchymal stem cells as an alternative cell model. 24
3.5 Sonogenetic stimulation of cbMSCs stably expressing Prestin(NT, NS) and pNFAT-hTRAIL. 26
3.6 Sonogenetic stimulation of specific gene expression in engineered 293T cells. 27
3.7 Transient transfection of 293T cells improves the sonogenetic induction of NFAT signaling. 29
Chapter 4. Discussion 31
4.1 Summary of current findings 31
4.2 The possible reasons for poor induction of NFAT signaling 32
4.3 The possible reasons to explain why Prestin(NT, NS) expressing cells induced higher NFAT-driven gene levels. 36
4.4 Future studies 37
Reference 39
Figures 47

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