本研究的目的，在於結合生化檢驗技術檢測草履蟲在超音波照射下的建設性生物效應（例如助長、活化、增量等）反應機制，以使得超音波在生物科技方面的應用，能更多元而深入。有別於以往以數量統計的方式做為生物生長率變化的指標，本研究係以生化的方法檢測蛋白質總量的變化，並配合生物計數法做為生物體生長及反應的指標。
　　研究中以Rayleigh-Plesset的空孔振動理論，作為計算草履蟲試體共振頻率的基礎，以設定超音波照射頻率的範圍。採用BCA（Bicinchoninic Acid）法做為蛋白質定量的方法，觀察其蛋白質的濃度變化。並配合生物計數法的觀察，以瞭解在超音波聲場下草履蟲的生物反應機制，由實驗的結果發現蛋白質的濃度與草履蟲的數量呈高度正相關，因此可以利用蛋白質的濃度去推算草履蟲的數量。
　　根據理論值所獲得的共振頻率範圍為0.467∼1.27 MHz，經過實驗驗證，以25% Tone Burst的1 MHz超音波持續照射草履蟲5分鐘後，在第72小時可觀察到最多的草履蟲數量。與對照比較利用蛋白質濃度反推的結果為1.77倍，利用生物計數法則有1.68倍。此研究成果提供了生化方式以外的刺激助長技術，及建設性超音波的另一佐證。

The problem of the long-term proliferation of cells is a seminal one. It has always been a hot subject in biology. In this reach, it try to improve the growth rate of paramecium by ultrasound exposure. To perform the above-mentioned research, the oscillation of the paramecium in response to the ultrasound radiation is simulated using Rayleigh-Plesset’s bubble activation theory. The gas body activation theory is to calculate the resonant frequencies of the paramecium at different stages of its life.
In this research, it will study the activation of the paramecium division using the resonant and non-resonant frequency of the ultrasonic exposure. The observing images obtained from a microscopic camera can be made and recorded by a personal computer. The biological effects such as the hatching period, growth rate, etc., can be observed from the images. In the past, the physical measurement such as the counting method is the easiest way to observe the biological effects of ultrasound. However, it is not sufficiently to analyze the quantitative bioeffect of paramecium by taking counts. Therefore, utilizing the biochemical technique to assay the difference of specimen may be a good point of view. In this research, employing BCA (Bicinchoninic Acid) assay to inspect the protein variation attain a brand-new quantitative analysis. The results obtained from this research can be used to develop the related ultrasonic biotechnology.
By using the theory, the calculated resonance frequency of the paramecium vacuole is about 0.467~1.27 MHz. The maximum amount of paramecium was observed in 72th hours with 1 MHz ultrasound exposure for 5 minutes. It’s 1.77 times relative to the control.

目錄I
表目錄.V
圖目錄.VII
中文摘要XIV
英文摘要XV
第一章　導論 1
1.1前言 1
1.2文獻回顧 3
1.3研究目的 9
1.4草履蟲介紹 10
1.4.1原生動物 10
1.3.2草履蟲 12
第二章　理論分析 20
2.1空孔共振理論 20
2.1.1氣體活化作用 21
2.1.2內部空孔效應 21
2.1.3氣泡收縮時溫度及壓力的改變 22
2.1.4 Rayleigh-Plessent方程式 23
2.2藍伯特-比耳定律 24
2.3蛋白質定量技術 26
2.4生物統計分析 30
2.4.1集中趨勢及變異性 30
2.4.2簡單直線迴歸及相關 32
第三章　實驗方法及步驟 43
3.1實驗設定 43
3.1.1照射試體的選用及培養 44
3.1.2共振頻率計算 45
3.1.3照射系統設計 47
3.2實驗設備 49
3.3實驗步驟 54
3.3.1草履蟲濃度及培養環境的選定 55
3.3.2超音波參數及觀察點的選擇 56
3.3.3草履蟲濃度的檢定 57
第四章　實驗結果與討論 85
4.1決定母體及取樣數 85
4.2 超音波照射系統的評估 85
4.2.1 實驗水槽與照射試片內聲場強度分布量測 86
4.2.2草履蟲濃度與蛋白質濃度間的關係 88
4.2.3照射聲強的決定 88
4.2.4照射時溫度的控制 90
4.3實驗結果的統計 90
4.3.1表格資料 90
4.3.2圖形資料 92
4.4實驗結果的討論 96
第五章 結論與建議 124
5.1結論 124
5.2 建議事項與未來展望 126
參考文獻 129
附錄 A 133
A.1 空孔氣泡運動方程式[37] 133
A.2 Rayleigh-Plesset方程式[18] 135
A.2.1 無阻尼的線性震盪 136
A.2.2 共振頻率（ωr） 137
A.3 穩態阻尼振盪的共振頻率 137
附錄 B 142
B.1 BCA法中化學藥品的成分及含量 142
B.2 BCA法中所配製化學藥品的圖示 146

1. M. Dyson and J.B. Pond, “The Effect of Pulsed Ultrasound on Tissue Regeneration,” Physiotherapy, pp. 136-142, 1970.
2. E.N. Harvey, “Biological Aspects of Ultrasonics Waves, a General Survey,” Biol. Bull, 59, pp. 306-325, 1930.
3. P.R. Clark and C.R. Hill, “Physical and Chemical Aspects of Ultrasonic Disruption of Cells,” J. Acoust. Soc. Am., 47, pp. 649-653, 1970.
4. V. Ciaravino, H.G. Flynn, and M.W. Miller, “Pulsed Enhancement of Acoustic Cavitation: A Postulated Model, ” Ultrasound Med. Biol., 7, pp. 159-166, 1981a
5. V. Ciaravino, M.W. Miller, and E.L. Carstensen, “Pressure-mediated Reduction of Ultrasonically-induced Cell Lysis,” Radiat Res., 88, pp. 209-213, 1981b.
6. V. Ciaravino, M.W. Miller, and G.E. Kaufman, “The Effect of 1 MHz Ultrasound on the Proliferation of Synchronized Chinese Hamster V-79 Cells,” Ultrasound Med. Biol., 47, pp. 649-653, 1981c.
7. D.L. Miller and A.R. Williams, “Bubble Cycling as an Explanation of the Promotion of Ultrasonic Cavitation in a Rotating Tube Exposure System,” Ultrasound Med. Biol., 15, pp. 641-648, 1989.
8. I.J.C. Macintosh and D.A. Davey, “Chromosome Aberrations Induced by an Ultrasonic Fetal Pulse Detector,” Br. Med. J., 4, pp. 92-93, 1970.
9. D. Liebeskind, R. Bases, F. Mendez, F. Elequin, and M. Koenigsberg, “Sister Chromatid Exchanges in Human Lymphocytes after Exposure to Diagnostic Ultrasound,” Science, 205, pp. 1273-1275, 1979a.
10. D.L. Miller, J.A. Reese, and M.W. Frazier, “Single Strand DNA Breaks in Human Leukocytes Induced by Ultrasound in Vitro,” Ultrasound Med. Biol., 15, pp. 765-771, 1989.
11. D.L. Miller, R.M. Thomas, and M.E. Frazier, “Single Strand Breaks in CHO Cell DNA Induced by Ultrasonic Cavitation in Vitro,” Ultrasound Med. Biol., 17, pp. 401-406, 1991a.
12. D.L. Miller, R.M. Thomas, and M.E. Frazier, “Ultrasonic Cavitation Indirectly Induces Single Strand Breaks in DNA of Viable Cells in vitro by the Action of Residual Hydrogen Peroxide,” Ultrasound Med. Biol., 17, pp. 729-735, 1991b.
13. D.L. Miller, R.M. Thomas, and R.L. Buschbom, “Comet Assay Reveals DNA Strand Breaks Induced by Ultrasonic Cavitation in Vitro,” Ultrasound Med. Biol., 21, pp. 841-848, 1995.
14. A.J. Mortimer and M. Dyson, “The Effect of Therapeutic Ultrasound on Calcium Uptake in Fibroblasts,” Ultrasound Med. Biol., 14, pp. 499-506, 1988.
15. G.E. Kaufman, “Mutagenicity of Ultrasound in Cultured Mammalian Cells,” Ultrasound Med. Biol., 11, pp. 497-501, 1985.
16. National Council on Radiation Protection and Measurements (NCRP), “Biological Effects of Ultrasound: Mechanism and Clinical Implications,” NCRP Report No. 74, Bethesda, MD: NCRP,1983.
17. National Council on Radiation Protection and Measurements (NCRP), “Exposure Criteria for Medical Diagnostic Ultrasound: I. Criteria based on Thermal Mechanisms,” NCRP Report No. 113, Bethesda, MD: NCRP,1992.
18. E.A. Neppiras, “Acoustic Cavitation,” Physics Reports-Review Section of Physics Letters, 61(3), pp. 159-251, 1980.
19. M. Dyson, “Non-thermal Cellular Effects of Ultrasound,” Br. J. Cancer Suppl., 45(5), pp. 165-171, 1982.
20. J.W. Ellwart, H. Brettel ,and L.O. Kober, “Cell Membrane Damage by Ultrasound at Different Cell Concentrations,” Ultrasound Med. Biol., 14(1), pp. 43-50, 1979.
21. Y.P. Yip, C. Capriotti, S.G. Norbash, M.S. Rosenthal, and J.W. Yip, “Ultrasound Effects on cell Proliferation and Migration of Chick Motoneurons,” Ultrasound Med. Biol., 17(1), pp. 55-63, 1991.
22. G. Cum, G. Galli, R. Gallo, and A. Spadaro, “Role of Frequency in the Ultrasonic Activation of Chemical Reaction,” Ultrasonics, 30(4), pp. 267-270, 1992.
23. T. Feigl, B. Völklein, H. Iro, C. Ell, and T. Schneider, “Biophysical Effects of High-Energy Pulsed Ultrasound on Human Cells,” Ultrasound Med. Biol., 22(9), pp. 1267-1275, 1996.
24. S.B. Barnett, H.D. Rott, G.R.T. Haar, M.C. Ziskin, and K. Maeda, “The Sensitivity of Biological Tissue to Ultrasound,” Ultrasound Med. Biol., 23(6), pp. 805-812, 1997.
25. J. Parvizi, C.C. Wu, D.G. Lewallen, J.F. Greenleaf, and M.E. Bolander, “Low-Intensity Ultrasound Stimulates Proteoglycan Synthesis in Rat Chondrocytes by Increasing Aggrecan Gene Expression,” J. Orthop. Res., 17(4), pp. 488-494, 1999.
26. K. Naruse, Y. Mikuni-Takagaki, Y. Azuma, M. Ito, T. Oota, and K. Kameyama, M. Itoman, “Anabolic Response of Mouse Bone-Marrow-Derived Stromal Cell Clone ST2 Cells to Low-Intensity Pulsed Ultrasound,” Biochem. Biophys. Res. Commun., 268(1), pp. 216-220, 2000.
27. S. Takikawa, N. Matsui, T. Kokubu, M. Tsunoda, H. Fujika, K. Mizuno, and Y. Azuma, “Low-Intensity Pulsed Ultrasound Initiates Bone Healing in Rat Nonunion Fracture Model,” J. Ultrasound Med., 20(3), pp. 197-205, 2001.
28. J. Harle, V. Salih, F. Mayia, J.C. Knowles, and I. Olsen, “Effects of Ultrasound on the Growth and Function of Bone and Periodontal Ligament Cells In Vitro,” Ultrasound Med. Biol., 27(4), pp. 579-586, 2001.
29. G.P. Saborio, B. Permanne, and C. Soto, “Sensitive Detection of Pathological Prion Protein by Cyclic Amplification of Protein Misfolding,” Nature, 411, pp. 810-813, 2001.
30. 楊旭光，張國賢，水下反沾附技術，海下技術季刊，第五卷第二期，pp. 47-50，民國84年6月。
31. S.K. Yang and Y.C. Huang, “The Activation of Growth in Plant Roots by Ultrasound Exposure,” Biomedical Engineering: Applications, Basis and Communications, 12(3), pp. 53-58, 2000.
32. S.K. Yang and Y.C. Huang, “Biological Effects of Paramecium in Diffused Ultrasonic Fields,” Ultrasonics, 39, pp.525-531, 2002.
33. 編著者：梁象秋，方紀祖，楊和荃，水生生物學（AQUATIC BIOLOGY），水產出版社，pp. 240-241，民國八十七年二月一日初版一刷。
34. Edited by H.D. Görtz, Paramecium, Spring-Verlag Berlin Heidelberg Germany, 1988.
35. Wichterman, Ralph, The biology of Paramecium, New York, Blakiston, c1953
36. Edited by Janet Kurjan, Barry L. Taylor, Signal Transduction : Prokaryotic and Simple Eukaryotic Systems, San Diego : Academic Press, 1993c.
37. D.L. Miller, “A Review of the Ultrasonic Bioeffectiveness of Microsonation, Gas-body Activation, and Related Cavitation-like Phenomena,” Ultrasound Med. Biol., 13, pp. 443-470, 1987.
38. W. Lauterborn, “Numerical Investigation of Nonlinear Oscillations of Gas Bubbles in Liquids,” J. Acoust. Soc. Am., 59(2), pp. 283-293, 1976.
39. D. Calloway, “Beer-Lambert Law,” J. Chem. Educ., 74(7), pp. 744, July 1997.
40. A.G. Gornall, C.J. Bardawill, M.M. David, “Determination of Serum Proteins by Means of the Biuret Reaction,” J. Biol. Chem., 177, pp. 751-766, 1949.
41. Lowry, N.J Rosebrough, A.L. Farr, and R.J. Randall, “Protein Measurement with the Folin-Phenol Reagents,” J. Biol. Chem., 193, pp. 265-275, 1951.
42. M.M. Bradford, “A Rapid and Sensitive Method for the Quantification of Microgram Quantities of Protein Utilizing the Principle of Protein-dye Binding,” Anal. Biochem., 72, pp.248-254, 1976.
43. P.K. Smith, “Measurement of Protein Using Bicinchoninic Acid,” Anal. Biochem., 150, pp. 76-85, 1985.
44. K. Wiechelman, R. Braun, and J. Fitzpatrick, “Investigation of the Bicinchoninic Acid Protein Assay: Identification of the Groups Responsible for Color Formation,” Anal. Biochem., 175, pp. 231-237, 1988.
45. R. Kessler and D. Fanestil, “Interference by Lipids in the Determination of Protein Using Bicinchoninic Acid,” Anal. Biochem., 159, pp. 138-42, 1986.
46. R. Brown, K. Jarvis, and K. Hyland, “Protein Measurement Using Bicinchoninic Acid: Elimination of Interfering Substances,” Anal. Biochem., 180, pp.136-9, 1989.
47. C.V. Sapan, R.L. Lundblad, and N.C. Price, “Review: Colorimetric Protein Assay Techniques,” Biotechnol. Appl. Biochem., 29, pp. 99-108, 1999.
48. 楊志良，生物統計學新論，九州，1996。
49. 戴正，江淑瓊，生物醫學統計概論，翰蘆，2000年1月。
50. B. Martin, An Introduction to Medical Statistics 3rd, Oxford, 2000.
51. M.W. Miller, D.L. Miller, and A.A. Brayman, “A Review of in vitro Bioeffects of Inertial Ultrasonic Cavitation from a Mechanistic Perspective,” Ultrasound in Med. & Biol., 22(9), pp. 1131-1154, 1996.
52. W.L. Nyborg and M.C. Ziskin, Biological Effects of Ultrasound, Churchill Livingstone, pp. 9-11, 36-38, 1985.
53. 楊旭光，「超音波生物效應：草履蟲在不同激振波型下的變化機制」，國科會專題計畫，NSC-90-2212-E-110-024, 民國91年8月。