- 學位論文
超音波微氣泡基因轉殖的物理參數對內皮起源細胞轉殖效應及細胞存活之影響
Effects of Physical Parameters on Efficiency and Cell Viability of Ultrasonic Microbubble-mediated Gene Delivery in Endothelial Progenitor Cells
摘要
本研究的目的在探討利用超音波微氣泡基因轉殖法(ultrasonic Microbubble-mediated transfection, UMT)於報導基因傳遞(reporter gene delivery)之效應，在基因傳遞實驗中利用不同的物理參數觀察其蛋白質表現量及趨勢，並根據各物理參數的實驗結果，得到一系列的物理參數作為未來超音波微氣泡基因轉殖法最適合的工作參數。超音波微氣泡基因轉殖法在基因傳遞中已被證實是一可行且安全的方法，但應用在內皮起源細胞(Endothelial Progenitor Cells, EPCs)上的反應及傳遞效果仍不清楚。內皮起源細胞為一下游血管幹細胞，能夠有效的幫助體內組織缺血時的血管新生(angiogenesis)並減少組織傷害的擴大。本實驗將以體外細胞實驗(in vitro)配合超音波系統，將Luciferase pLuc-N3 DNA(5.6 kb, cytomegalovirus (CMV) promoter)報導基因傳遞至EPCs內，來測試對基因表現量及細胞存活率之影響。實驗結果顯示，蛋白質表現量(protein expression)及細胞存活數(cell viability)會隨著基因轉殖前細胞培養時間長短、質體DNA(plasmid DNA, pDNA)濃度、微氣泡濃度、轉殖溫度、超音波聲壓強度、超音波工作週期(duty cycle, DC)及超音波曝露時間(ultrasound exposure time)產生變化，最後根據各實驗結果，得到一相對合適的工作參數條件－基因轉殖前細胞培養時間：72小時、質體DNA濃度：15 μg/ml、微氣泡濃度：3 % (v/v)、轉殖溫度：37℃、超音波聲壓強度：1 W/cm2、超音波工作週期：10 %與超音波曝露時間：30秒。 最終實驗結果顯示，使用新的物理參數進行超音波微氣泡基因轉殖法，其蛋白質表現量能有效提高3.5倍以上，在細胞存活數的表現上仍有60 % ~ 70 %的存活率，於統計分析上比較後也具有顯著差異(p < 0.001)，顯示其基因傳遞的效率有很好的表現結果。此系列工作參數將作為未來超音波微氣泡基因轉殖應用在EPCs生體研究(in vivo)上的基礎參數。
並列摘要
Ultrasonic microbubble-mediated gene delivery has shown to be a feasible method in gene delivery. Endothelial progenitor cells (EPCs) have emerged as potentially useful substrates for neovascularization, tissue repair, and bioengineering. The responsiveness of different physical parameters of ultrasonic microbubble transfection (UMT) on EPCs remained unclear. EPCs originated from porcine peripheral blood were perfused with solution containing luciferase reporter gene and suspended microbubbles (1.5 ~ 2.5 μm in diameter) followed by exposure to therapeutic ultrasound (US) with different transfection parameters. The physical parameters included US exposure timing (24, 48 and 72 hours after cell seeding), DNA concentration (5, 15 and 30 μg/ml), microubble concentration (3 % and 10 % (v/v)), transfection temperature (23, 30, 37 and 45℃), and ultrasonic parameters (intensity, duty cycle and US exposure time). Luciferase gene expression was evaluated 48 hours post transfection. In parallel, a series of ultrasonic parameters were conducted to investigate the delivery of pDNA into EPCs. In the presence of microbubbles, the relatively low intensity of ultrasonic parameters of 1 W/cm2, duty cycle 5 % and exposure time 30 seconds, which yielded a relatively high luciferase expression up to 200 LU/cell (n = 4 ~ 7, p < 0.001 vs control), were selected for the gene delivery. The use of physical transfection factors of ultrasound exposure timing (72 hours after cell seeding), DNA concentration (15 μg/ml), microubble concentration (3 % (v/v)), and temperature (37℃) can significantly improve gene expression (all p < 0.05). Cell viability post transfection is 60 % ~ 70 % in average (all p > 0.05 vs individual control). UMT successfully delivers reporter gene into porcine EPCs by using different sets of US parameters. Among physical factors, ultrasound exposure timing (72 hours after cell seeding, P < 0.001), DNA concentration (15 μg/ml), microubble concentration (3 % (v/v)), temperature (37℃), and ultrasonic parameters (intensity 1 W/cm2, DC 10 %, and exposure time 30 sec) are optimal for ultrasonic microbubble-mediated gene delivery into EPCs. By using the optimized US parameters, the luciferase protein expression can be enhanced up to 3.5 fold (P < 0.001), compared to the initial parameters. Our findings provide a practical basis to achieve relatively high delivery efficiency together with a cell viability more than 60 % ~ 70 %.