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研究生(外文):Shin-Yu Fang
論文名稱(外文):The Effect of Ultrasound Acoustic Parameters on the Gene Transfection Efficiency: in Vitro Study
指導教授(外文):Win-Li Lin
外文關鍵詞:pulse repetition frequencyacoustic parameterultrasoundgene transfection
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如何發展正確、安全及有效的基因傳送方法在超音波基因治療中一直是主要的挑戰,本篇論文主旨在於找出最佳化的超音波聲參數使基因轉殖效率以及細胞存活率皆能增加。本篇論文中共討論超音波能量劑量(energy dose),超音波施打策略以及超音波脈衝重複頻率(pulse repetition frequency)三種超音波聲參數;一般而言,超音波能量劑量越高,轉殖效率越高,但死亡率也相對提高,在本研究中發現,對於子宮頸癌細胞(HeLa cell)而言,55 W(4.8 W/cm2) ~ 60 W (5.3 W/cm2)似乎是最好的超音波能量強度,而超音波照射時間需超過15秒,太高或太低的超音波劑量皆導致低轉殖效率;另外於“on-off”的超音波施打策略中,我們期待細胞於超音波能量“off”期能有足夠時間修補因超音波所造成的傷害,進而提高細胞存活率,而於下一次超音波能量“on”的期間則可進行下一階段的基因轉殖,因此我們測試在同樣的總“on”時間下(20秒),兩階段的超音波照射(各10秒)間隔不同的“off”時間(3秒、10秒、30秒、1分鐘與10分鐘)的基因轉殖效果,然而實驗結果證實此策略未能增加基因轉殖效率及細胞存活率,第二階段的超音波施打未能有基因轉殖的功能,推測其原因可能為超音波能量“off”期皆太長,細胞可能已產生對外來刺激的抵抗反應。我們進一步探討超音波脈衝重複頻率(PRF)對轉殖效率的影響,實驗結果發現當超音波工作週期(duty cycle)為50%且脈衝重複頻率低於2 Hz時,細胞擁有高轉殖效率,但會造成高溫升及高死亡率,當超音波工作週期(duty cycle)為50%且脈衝重複頻率高於1000 Hz時,轉殖效率明顯開始下降,而當超音波工作週期(duty cycle)為50%且脈衝重複頻率高於4000 Hz時,細胞雖然有高存活率及低溫升,但轉殖效率卻為0%,因此當超音波工作週期(duty cycle)為50%時,最佳脈衝重複頻率應為20 Hz 到1000 Hz之間;另外由實驗結果中可發現,當超音波每一次的施打時間與暫停時間比例(on-time/off-time)小於0.1時,轉殖效率急速下降(可降到0%),另外回顧兩階段”on-off”策略,亦可發現其施打時間與休息時間比例皆小於0.1,因此第二次超音波施打並未有轉殖效率,故總結而言,建議超音波每一次的施打時間與暫停時間比例(on-time/off-time)需大於0.1。
Ultrasound bioeffects highly depend on the exposure parameters such as sonication time, burst length, pressure amplitude, intensity and the pulse repetition frequency. The optimal parameters for ultrasound-mediated gene transfection have not been systematically determined. On this ground, we studied the effect of different acoustic parameters with the intention to find out the optimal parameters to increase both the transfection efficiency and the cell survival. We modified the following acoustic parameters: the energy dose (including the ultrasound intensity and ultrasound exposure time), the “off” time, and the pulse repetition frequency (PRF).
It was found that the optimal ultrasound power (intensity) would be between 55 W (4.8 W/cm2) and 60 W (5.3 W/cm2) while the ultrasound duty cycle was 50% for a total 20-sec exposure (PRF=200 Hz, 2500 cycles). The energy dose of 55 W-15 sec would be the threshold for gene transfection.
Although the ultrasound exposure could increase the transfection efficiency, the results of the on-off strategy contrasted to our intention. A short time break (3 sec, 10 sec, 30 sec, 1 min, or 10 min) would not increase the transfection efficiency or cell survival (however it can avoid extremely high temperature rise). One reasonable explanation might be the off-time was too long. Cells might have already induced some shock-resistive or stress-resistive mechanism.
On the other hand, we found that (1) the transfection efficiency declines rapidly while the PRF was higher than 1000 Hz. (2) When the PRF was lower than 2 Hz (the exposure on-time was longer than 250 ms), there was low cell survival probably because of the high temperature rise, although the transfection efficiency was relatively high. (3) The optimal PRF would be between 20 Hz and 1000 Hz. (4) While the PRF was between 20 Hz and 1000 Hz, a better transfection would be obtained when the “on” time to the “off” time ratio was greater than 0.1.
Overall, ultrasound-assisted gene delivery can improve the efficiency of nonviral gene delivery. Further studies for its mechanisms will be our future work.
Abstract III
Contents IV
List of Figures VI
List of Tables X
1. Introduction 1
2. Material and Methods 5
2.1 Cell culture and plasmid preparation 5
2.2 Ultrasound setup 7
2.2.1 Setup design 7
2.2.2 Pressure measurement 10
2.3 In vitro experiments 11
2.3.1 Preliminary experiments 11
2.3.2 Acoustic parameters 12
2.3.3 Hyperthermia experiments 17
2.4 Statistical analysis 18
3. Results 19
3.1 Pressure field 19
3.2 Preliminary experiments 21
3.3 Acoustic parameters 24
3.3.1 Energy dose 24
3.3.2 On-off strategy 33
3.3.3 Pulse repetition frequency (PRF) 41
3.4 Hyperthermia experiments 62
4. Discussions 64
5. Conclusions 68
References 69
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