靜磁場曝照對超音波微氣泡基因傳遞之增強效應-體外細胞實驗

The Enhanced Effect of Static Magnetic Fields on Ultrasound-Microbubble Medicated Gene Delivery In Vitro

簡瑋文(Wei-Wen Chien)

指導教授 ： 呂志誠 ； 蘇正煌

國立臺北科技大學/機電學院/機電整合研究所/碩士(2010年)

摘要

本研究目的在探討施加不同工作週期(duty cycle, DC)超音波能量並結合靜磁場(static magnetic fields, SMFs)作用，進行超音波微氣泡轉染於基因傳遞效能之研究。利用自行建構一套適用於體外實驗(in vitro)的超音波基因傳遞系統，以超音波微氣泡輔助所產生的穴蝕效應(cavitation)增加細胞膜通透性並促進報導基因pGL3 luciferase進入細胞內，以提高基因傳遞效能。本研究利用銣鐵硼(Nd-Fe-B)磁鐵產生之靜磁場環境，讓細胞於培養過程中曝照靜磁場48小時，實驗中施以不同工作週期及聲壓強度的超音波能量，並加入Artison商用微氣泡(microbubble, MB)作為輔助，將pGL3 luciferase基因傳遞至HAEC (human aortic endothelial cells, 人類主動脈內皮細胞)，觀察各参數間對基因表現量與細胞存活率之影響。實驗結果顯示相對於對照組(control)，單純施加超音波所得到的基因表現量並無明顯增加，但加入微氣泡輔助後則可大幅提升基因傳遞效能，且基因傳遞效能會隨著超音波工作週期的提升而增加。超音波微氣泡轉染配合靜磁場作用之基因表現量皆高於未受靜磁場作用，尤其當超音波聲強於0.3 MI和0.5 MI時，受靜磁場作用後的基因表現量分別增加2.2和1.8倍，於統計分析上有顯著差異；顯示細胞受靜磁場作用後，有助於提升基因傳遞效能。此外，超音波微氣泡輔助有(無)配合靜磁場作用之細胞存活數與對照組相比約減少15 - 25 %，表示微氣泡受超音波作用產生的穴蝕效應可能造成部分細胞死亡，而靜磁場的作用並不會影響細胞數目。

關鍵字

基因傳遞 ； 穴蝕效應 ； 報導基因 ； 靜磁場 ； 微氣泡

並列摘要

The purpose of this study is to envestigate the enhanced impact of different ultrasound duty cycles (DC) combined with static magnetic fields (SMFs) upon the ultrasound-microbubble mediated transfection on gene delivery. By using microbubbles that can generate cavitation to increase cell membrane permeability, and promote the pGL3 luciferase report gene transported into cells for improved gene transfer efficiency, an ultrasound-assisted system was established for in vitro gene delivery experiments. The target cells were exposed to the static magnetic fields produced by Nd-Fe-B permanent magnets in an incubator for 48 hours. Experiments employing the pGL3 luciferase report gene to be delivered to HAEC (human aortic endothelial cells) for gene expression and cell viability were carried out with specific parametric sets of ultrasound duty cycle and acoustic intensity and Artison® microbubbles. Our experimental results conclude that the gene expression is not significantly increased when using ultrasound alone, but it is significantly increased (p<0.001) when using ultrasound-mediated microbubble compared to the control set. Moreover, the gene transfer efficiency will accordingly improve as the ultrasound duty cycle was increased. Most importantly, the intensity of gene expression is proven to increase when using ultrasound-mediated microbubble with SMFs compared to ultrasound-mediated microbubbles; especially while ultrasound acoustic intensity is between 0.3 and 0.5 MI, the gene expression is increased 2.2 and 1.8 times, respectively. The experimental results also demonstrate that when cells are exposed to SMFs, it really helps to improve the gene transfer efficiency. In addition, the cell viability decreases about 15-25 % when using ultrasound-mediated microbubble with or without SMFs compared to the control set, which means that ultrasonic assisted microbubbles can generate significant cavitation and cause cell casualty.

並列關鍵字

gene delivery ； cavitation ； reporter gene ； static magnetic fields ； microbubble ； human aortic endothelial cell

參考文獻

[39] 白啟宏，靜磁場與超音波微氣泡轉染對於基因傳遞影響研究，碩士論文，國立台北科技大學機電整合所，台北，2009。

[1] A. Halama, M. Kuliñski, T. Librowski, S. Lochyñski, “Polymer-based non-viral gene delivery as a concept for the treatment of cancer,” Pharmacol Reports, vol. 61, no. 6, 2009, pp. 993-999.

[3] M. Mata, S. Hao, D. J. Fink, “Applications of gene therapy to the treatment of chronic pain,” Curr Gene Therapy, vol. 8, 2008.

[4] R. J. Connolly, G. A. Lopez, A. M. Hoff, M. J. Jaroszeski, “Characterization of plasma mediated molecular delivery to cells in vitro,” International Journal of Pharmaceutics, vol. 389, 2010, pp. 53–57.

[5] L. C. Phillips. A. L. Klibanov, D. K. Bowles, M. Ragosta, J. A. Hossack, B. R. Wamhoff, “Focused in vivo delivery of plasmid DNA to the porcine vascular wall via intravascular ultrasound destruction of microbubbles,” Journal of Vascular Research, vol. 47, no. 3, 2010, pp. 270-274.