A Study on Acoustic Cavitation Assisted Gene Delivery

基因治療是目前極具潛力的治療模式，其定義為”修復可能致病基因的技術”。基因治療的關鍵為基因傳遞（又稱為基因轉殖），可藉由許多方法達成，本研究使用之方法為利用超音波與超音波對比劑誘發穴蝕效應，以達成基因傳遞。穴蝕效應為微氣泡於聲場中受到壓力變化而脹縮與破裂的過程，由超音波產生的穴蝕效應在非熱治療領域上之優點如下：由於超音波具有非侵入性及深處聚焦之能力，因此應用於治療較安全可靠；且其亦為發展完善的造影方法，故可將治療與影像結合。雖然以超音波穴蝕效應進行基因傳遞已有許多成功的案例，且其機制目前認為是穴蝕效應導致細胞膜通透性增加，但對於穴蝕效應與基因傳遞間的量化關係仍然不清楚，故本研究的目的即是量化基因傳遞效率/細胞存活率與穴蝕效應之關係。本研究使用1MHz超音波探頭與商用對比劑Levovist®來誘發穴蝕效應，並以HeLa（子宮頸癌）細胞與標訂FITC染劑的DNA進行基因傳遞實驗。研究中使用超音波發射聲壓、對比劑濃度、超音波發射週數作為改變的參數，先有效地誘發與偵測穴蝕效應，並以慣性穴蝕劑量定量微氣泡破裂的程度，再進行基因傳遞實驗，於螢光顯微鏡下計數基因傳遞率與細胞存活率，最後以基因傳遞結果為縱軸，慣性穴蝕劑量為橫軸，將兩者的間之量化關係畫出，並比較不同超音波參數的結果之相關性。實驗結果發現，穴蝕效應（微氣泡破裂）的趨勢會隨發射聲壓增強、微氣泡濃度提高、發射週數增加而明顯升高，表示本研究已可有效地誘發與偵測穴蝕效應。基因傳遞實驗方面，單一變因下的基因傳遞率/細胞存活率對於慣性穴蝕劑量具高度相關性。若結合由不同超音波參數得到的基因傳遞率/細胞存活率對於慣性穴蝕量圖，其相關係數R2亦達0.95與0.84，代表基因傳遞結果與慣性穴蝕劑量密切相關。此結果表示同一架構下不論改變的超音波參數為何，穴蝕效應與基因傳遞結果間存在一固定的函數關係，即有可能運用穴蝕效應的實驗結果來預測基因傳遞效率與細胞存活率。Gene therapy is a promising therapeutic technique, and it is defined as correcting defective genes responsible for disease development. The key of gene therapy is gene delivery (also known as transfection). In this research, ultrasound and ultrasound contrast agents are used to induce cavitation, and gene delivery is assisted with cavitation effect. Cavitation is the process of microbubble formation, resonance, and destruction. When microbubbles experience time-varying acoustic field, cavitation may occur. Cavitation effects induced by ultrasound have been found beneficial in the non-thermal treatment, and the advantages of ultrasound are non-invasiveness, focusing and real-time imaging capacities. Although quantitative success of acoustic cavitation assisted gene delivery has been reported in the literature, the relations between gene delivery rate and cavitation effect are still unknown. In this research, methods for effective induction of acoustic cavitation are first established. Then, the quantitatively relations of gene delivery and cavitation dose are analyzed. A 1MHz ultrasound transducer and the commercial ultrasound contrast agent Levovist® are used for cavitation induction. HeLa cells and FITC labeled short sequence DNA are used in the gene delivery experiments. The parameters under investigation are acoustic pressure, microbubble concentration and ultrasound pulse length. The effect of cavitation (microbubble destruction) is quantitatively analyzed using the inertial cavitation dose (ICD) method. After gene delivery experiments, the gene delivery rate and cell viability are calculated under the fluorescent microscope. The curves of gene delivery rate (and cell viability) versus ICD are plotted, and the relations between gene delivery rate/viability and cavitation are examined. The results show that ICD increases with the acoustic pressure, microbubble concentration and pulse length. In the gene delivery experiments, there are high correlations between gene delivery rate (or cell viability) and ICD. Combining the results of gene delivery rate (or cell viability rate) versus ICD of the three parameters, high correlations also exist between gene delivery results and ICD, indicating that gene delivery results may be potentially predicted using cavitation measurements before gene delivery experiments.第一章 緒論 1 1.1基因治療 1 1.2超音波治療之演進 4 1.3穴蝕效應 6 1.4超音波於基因傳遞之應用 8 1.5研究動機與目標 10 1.6論文架構 12 第二章 實驗材料與方法 13 2.1穴蝕效應實驗—誘發與偵測 13 2.1.1超音波對比劑 13 2.1.2實驗系統架構 15 2.1.3穴蝕效應之誘發 17 2.1.3.1發射聲場強度 17 2.1.3.2微氣泡濃度 17 2.1.3.3發射週數 18 2.1.4穴蝕效應之偵測 19 2.1.4.1 B-mode影像觀察法 19 2.1.4.2穴蝕效應之定量表示—Inertial Cavitation Dose 19 2.2基因傳遞實驗 22 2.2.1細胞株 22 2.2.2 DNA 23 2.2.3系統架構與實驗流程 24 2.2.3.1基因傳遞率量測法 24 2.2.3.2細胞存活率量測法 24 2.2.4實驗參數 26 2.2.5基因傳遞率/細胞存活率與穴蝕效應關聯之建立 28 第三章 實驗結果 29 3.1穴蝕效應實驗結果 29 3.1.1 B-mode影像觀察法 29 3.1.2發射聲壓 vs. 穴蝕效應 30 3.1.3微氣泡濃度vs. 穴蝕效應 34 3.1.4發射週數 vs. 穴蝕效應 38 3.2基因傳遞實驗結果 40 3.2.1螢光顯微鏡與共軛焦顯微鏡觀察結果 40 3.2.2發射聲壓 vs. 基因傳遞率與細胞存活率 43 3.2.3微氣泡濃度 vs. 基因傳遞率與細胞存活率 46 3.2.4發射週數 vs. 基因傳遞率與細胞存活率 49 3.3穴蝕效應 vs. 基因傳遞率與細胞存活率 50 第四章 分析與討論 52 4.1穴蝕效應之測與定量 52 4.2基因傳遞實驗結果分析 57 4.3穴蝕效應與基因傳遞率、細胞存活率之關聯 59 4.4本研究之應用 61 第五章 結論與未來工作 62 第六章 參考文獻 6