中文摘要
本論文的目的在於研究單細胞原生動物—草履蟲(Paramecium)，經由不同的超音波頻率以及各種聲學參數的設定來進行超音波照射，其生物或行為上所可能產生的變化機制，以呈現出超音波生物效應在單細胞生物研究上的完整面貌。以往超音波應用於生物、食品或醫藥等相關產業時，多半使用較高強度的超音波，產生強大的熱效應或劇烈的空蝕效應來破壞細胞與組織；然而這些超音波相關技術雖然行之有年，卻因為對於各種聲學參數與生物間的相對關係並不瞭解，亦或是相關理論基礎的不完備，造成這些超音波技術在使用上只知其然不知所以然，也就讓這項技術看似實用，事實上卻潛藏許多的危機。因為當越來越多的醫療儀器使用超音波來進行相關的治療或復健時，這項技術所可能造成的破壞效應便不是我們所樂見的。
也因為這項議題的嚴重性，所以對於超音波生物效應的相關研究便刻不容緩。本論文經由多項文獻的回顧中發現，在過去對超音波生物效應的研究上，多半是以高強度的超音波穿透組織或細胞，誘發多細胞的組織或細胞內所含的氣室與空氣管路產生劇烈振動，導致組織中接近氣室或空氣管路的細胞，因為機械剪切力而造成細胞膜或細胞壁的破壞，因而產生再生率的降低或相對成長率的突降。以上的過程在文獻中稱之為穩態空孔效應。這些研究使得超音波在生物效應的研究及應用上，被定位為破壞與消除的機制，但對於超音波所致的建設性生物效應（例如助長、增量等），卻往往忽略。
本論文即針對上述問題，並考慮多細胞生物在接受超音波照射時，其內部各組織或結構常互相關連，導致所衍生的生物效應往往極為複雜，而不易觀察出生物效應產生的真正機制，因此特別考量以生命最原始形態的單細胞動物—草履蟲為試體，希望能更清楚呈現超音波對生物效應的影響。本研究並以雷利(Rayleigh)氣泡振動理論模擬試體的結構特性，計算其共振頻率，以作為區分共振頻率與非共振頻率區之依據，並適度的安排照射時間與強度，作為超音波照射聲場的設定。而在生物效應的觀察上，則是將觀察影像經數位化處理後，進行照射前後的比較。其樣本統計的方法則以血球計數法與顯微鏡計數的方法互相搭配使用，以增加觀測之精度。此研究之結果，不僅可釐清超音波聲學參數中有關頻率、強度與照射時間在生物效應的相對關係，更可作為爾後超音波生物技術之研究基礎。

ABSTRACT
The effects of ultrasonic irradiation at different frequencies, i.e. 0.25, 0.5, 1, and 5 MHz, on the biological reaction of the single cell creature have been investigated. When multi-cell creature is exposed to ultrasound, this reaction will lead the biological effect becomes complex. Therefore, in this dissertation, a single cell creature is chosen to study the biological effects induced by ultrasound exposure. The paramecium, which possesses many features typical of higher-order animal cells, was considered as an appropriate choice for this study. The ability shown by ultrasound in promoting and/or accelerating many reactions has been shown to be a useful field. The resonant frequency of paramecium by using the ultrasound irradiation is an important parameter in this research. All other parameters being kept constant, it has been ascertained that an appropriate frequency value of ultrasound can be selected, capable of driving a biological reaction to its suitable yield. The oscillation of the cells in response to the ultrasound radiation is simulated using Rayleigh-Plesset’s bubble activation theory. The resonant frequency of the unicellular creature is then calculated. In the experiment, the resonant (0.5 and 1 MHz) and non-resonant (0.25 and 5 MHz) frequencies were employed.
The theoretical resonant frequency of the paramecium vacuole is among 0.5013~1.2703 MHz. In this thesis, the experiment included two different series. The exposure intensity is the major subject of the first experimental series to study the bioeffect of ultrasound. The waveform was set to the tone pulse mode, pulsing 1:1. The exposure duration was continued and maintained 5 minutes. For a given frequency, exposures of the paramecium were made over a range of intensities spanning 0 to 1.7 mW/cm2. The second experimental series was focused on the exposure duration of the ultrasound. The transducers were operating in a pulsed mode with two duty cycles of 1:1 and 1:9. All insonated samples were exposed to ultrasound with a spatial peak temporal peak intensity (SPTP) of 0.127 mW/cm2. The total “with signal” time was about 6 minutes in each trial. In addition, the control samples and the treated samples would be re-incubated up to the 96 hr. When the 1 MHz frequency of ultrasound was irradiated in the samples, there was about 24% inhibition rate and 30% enhancement rate in the first experimental series. The 0.5 MHz frequency of ultrasound, which approaches to the resonant frequency range, also appeared the inhibitive and beneficial effect. In the second experimental series, the relative growth number was about 32.4% higher than that of unexposed sample. The inhibition or enhancement growth conditions did not appear apparently during irradiation the non-resonant frequency of ultrasound. Moreover, experimental evidence suggests that the sustaining growth effect can be expected, when the irradiation time is divided into parts.

CONTENTS

Contents……………………………………………………………………….....I
List of Figures…………………………………………………………………...II
List of Tables…………………………………………………………………....V
中文摘要………………………………………………………………………...VI
Abstract……………………………………………………………………….VIII
Chapter 1. Introduction…………………………..….………...…………………1
1.1 Algae or Plant Tissues………………………………….………………4
1.2 Insect Larvae………………………………………………..………….8
1.3 Cell Suspensions……………………………………..……….……..…9
1.4 Protozoan………………………………………………………...……15
1.4.1 Cellular Structure and Function…………………………………...16
1.4.1.1 Digestion………………………………………………………16
1.4.1.2 Respiration………………………………………………….…17
1.4.1.3 Reproduction…………………………………………………..17
1.5 Ciliophora…..………...………………………….……………………18
1.6 Paramecium………………...…………………………………………18
1.6.1 Movement…………..…….…………….…………………………20
1.6.2 Feeding………………………….………….……………….……..20
1.6.3 Reproduction…………..………….……………………..…..…….21
Chapter 2. Theory………………………………………………………………31
Chapter 3. Methods…………………………………………………………….41
3.1 Cell culture and proliferation…………………..……..………………..41
3.2 The exposure procedure………………………………………………..42
Chapter 4. Results and discussions……………………………………………..53
4.1 Exposure intensity test…………………………………………..……..53
4.2 Exposure duration test……………………………………….………...58
Chapter 5. Conclusion and future works………………………….……………86
Reference……………………………………………………………………….88

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