臺灣博碩士論文加值系統

超音波可以作為一種非侵入式的治療策略，它能將機械波傳遞到軟組織以造成細胞內部的反應。因此超音波產生的機械效應、空穴效應或微流可視為一種胞外訊息，進而影響幹細胞的增生與分化能力。許多臨床與動物實驗中顯示低強度脈衝超音波能加速骨折癒合、促進骨生長，但鮮少研究顯示有關於低強度脈衝超音波對於間質幹細胞分化成心肌細胞之相關文獻。因此，本研究的目的是探討超音波刺激對於間質幹細胞分化成心肌細胞之影響。研究中建立一套超音波驅動系統來產生低功率超音波，並利用可調式穩壓電路來調整輸出功率。本研究透過體外實驗模式，針對間質幹細胞施予不同工作週期與聲功率的超音波刺激，並分析細胞增生與分化成心肌之程度。實驗採用Sprague-Dawley 大鼠之股骨萃取間質幹細胞，經流式細胞儀分析顯示間質幹細胞表現CD90 抗體標識出的表面抗原所佔比例約為97.48 %，而CD34 抗體並無表現，因此，實驗所萃取的間質幹細胞為高純度的。之後將第2 代的間質幹細胞植入75T flask 培養皿 (細胞數為105 cells/well)。待多數細胞貼附完全後再對細胞實施超音波照射。超音波驅動頻率為1MHz，探討的聲學參數包括聲功率 (145mW、304.5mW與449.5mW) 及脈衝長度 (工作週期100%及20%)，照射時間固定為10分鐘。在第0、7 與14 天分析間質幹細胞之分化情形。實驗結果顯示脈衝超音波(工作週期20%)對於細胞增生有正面的影響，並且在第14 天的最大細胞增生量，約為39% (聲功率449.5mW)；而連續超音波 (工作週期100%) 則會造成細胞數目的減少。但在細胞分化程度上，連續超音波與脈衝超音波在第7 天的細胞分化量約為32.6% ~ 51.9%，其分化結果皆有顯著的影響，且二者之間差異不大；而在第14 天時連續超音波對細胞分化的表現則明顯優於脈衝超音波，其最大細胞分化量約為23.6% (聲功率304.5mW)。本研究的成果證實了超音波刺激確實對間質幹細胞分化成心肌細胞造成影響。

Ultrasound has been shown to be a noninvasive treatment method. It
produces mechanical waves through soft tissues to change intracellular responses. Mechanical forces, cavitation and microstreaming accompanied with ultrasound exposure could serve as extracellular information, which may directly affect stem cell proliferation and differentiation. Many animal and clinical studies have shown that low-intensity pulsed ultrasound (LIPUS) can promote healing in the fracture model and enhance bone growth. However, less study investigates the effects of LIPUS stimulation on mesenchymal stem cells (MSCs). The purpose of this study is to investigate the effects of ultrasonic stimulation on the differentiation of MSCs into cardiomyocytes. In this study, we accomplished an ultrasonic exposure system to drive ultrasound transducers to generate LIPUS energy. An in-vitro study was employed to investigate the performance of ultrasound exposure on the differentiation of MSCs. Cells were isolated from the Femurs of Sprague-Dawley rats. Following flow cytometry analysis, MSCs express the expression of surface markers which CD90 expression is 97.48% and CD34 is not expressed, which demonstrates the high purity of the isolated MSCs. The MSCs of the second passage are cultured in a 75T-flask (cell numbers is 105 cells/well). And we use pre-culture method to let more MSCs to attach on the 75T-flask. Then cells received the exposure of a 1 MHz ultrasound 10 minutes/day. Acoustic parameters studied include power level (145mW, 304.5mW and 449.5mW) and duty cycle (20% and 100%) of LIPUS. Cell proliferation and specific protein markers of cardiomyocytes are examined. at 0th, 7th and 14th day after MSCs have received ultrasonic exposure. Results show that pulsed ultrasound (duty cycle 20%) has a positive effect on the increase of cell numbers, and the maximum cell proliferation is 39 % at 14th day (acoustic power 449.5mW). On the contrary, continuous ultrasound (duty cycle 100%) makes the cell number decreased. In addition, both continuous and pulsed ultrasound have significant effects on cellular differentiation at 32.6% ~ 51.9% at 7th days. At 14th day, continuous ultrasound shows a better cellular differentiation effects than pulsed ultrasound with a differentiation ratio of 23.6% (acoustic power 304.5mW). Base on the experimental results, it shows that ultrasonic stimulation could affect the differentiation of MSCs into cardiomyocytes.

誌謝 ......................................................... I
中文摘要 ............................................................ II
Abstract ................................................... IV
目錄 ........................................................ VI
圖目錄 ....................................................... X
表目錄 ..................................................... XIV
論文架構 .................................................... XV
第一章 緒論 .................................................. 1
1-1 心肌梗塞(Myocardial Infarction；MI) ...................... 1
1-2 組織工程 ............................................................. 2
1-3 治療性超音波之物理刺激 .................................... 3
1-3-1 超音波在細胞上之研究 .................................... 4
1-3-2 超音波在傷口癒合之研究 .................................. 7
1-3-3 超音波在神經修復之研究 .................................. 8
1-3-4 超音波在軟骨細胞上之研究 ................................ 8
1-3-5 超音波在治療癌症之研究 .................................. 9
1-4 研究目的 ................................................ 12
第二章 理論基礎 ............................................. 13
2-1 超音波概述 .............................................. 13
2-1-1 波動的基本原理 ........................................ 13
2-1-2 超音波換能器(transducer)與聲場 ......................... 18
2-1-3 超音波之生物效應 ...................................... 20
2-2 細胞 .................................................... 22
2-2-1 幹細胞 ................................................ 22
2-2-2 間質幹細胞............................................. 23
2-2-3 誘導間質幹細胞分化心肌細胞 .............................. 25
第三章 超音波刺激器之理論與設計 ............................... 26
3-1 架構設計 ................................................ 26
3-2 波寬調變電路 ............................................ 27
3-3 半橋式電路 .............................................. 30
3-4 功率調變 ................................................ 31
3-5 聲功率量測 .............................................. 32
3-6 超音波刺激器之模擬與實測結果 .............................. 34
3-6-1 波寬調變電路之模擬與實測 ............................... 34
3-6-2 半橋式電路之模擬與實測 ................................. 38
3-6-3 功率調變之模擬與實測 ................................... 40
3-6-4 聲功率量測系統 ........................................ 42
第四章 細胞刺激實驗 .......................................... 44
4-1 實驗架構 ................................................ 44
4-2 實驗方法 ................................................ 45
4-2-1 骨髓間質幹細胞之萃取 ................................... 45
4-2-2 骨髓間質幹細胞之培養 ................................... 45
4-2-3 骨髓間質幹細胞之誘導培養 ............................... 45
4-3 超音波刺激系統之架構 ..................................... 46
4-4 實驗設計 ................................................ 50
4-4-1 超音波穿透培養皿前後的功率大小差異 ...................... 50
4-4-2 不同工作週期(Duty cycle)與聲功率的超音波刺激細胞 ........ 50
4-5 分析方法 ................................................ 52
4-5-1 細胞形態 (morphology) 之分析 .......................... 52
4-5-2 細胞數目之計數 ........................................ 52
4-5-3 流式細胞儀(Flow cytometry)之分析 ....................... 54
4-5-4 統計分析 .............................................. 55
第五章 實驗結果 ............................................. 56
5-1 超音波穿透培養皿之聲功率量測結果........................... 56
5-2 超音波刺激細胞之結果 ..................................... 59
5-2-1 大鼠骨髓間質幹細胞之培養與鑑定分析 ...................... 59
5-2-2 不同聲功率的連續超音波對細胞刺激之結果 ................... 61
5-2-3 不同聲功率的脈衝超音波對細胞刺激之結果 ................... 67
5-2-4 連續超音波與脈衝超音波之結果比較 ........................ 74
第六章 結論與未來展望 ........................................ 80
6-1 結論 .................................................... 80
6-2 未來展望 ................................................ 82
參考文獻 .................................................... 83

[1] Available, "http://www.mohw.gov.tw/."
[2] 王錫崗, "人體生理學," 新文京開發, 2006.
[3] C. Becker and G. Jakse, "Stem Cells for Regeneration of Urological
Structures," European Urology, vol. 51, pp. 1217-1228, 2007.
[4] F. J. O''Brien, "Biomaterials & scaffolds for tissue engineering,"
Materials Today, vol. 14, pp. 88-95, 2011.
[5] G. ter Haar, "Therapeutic ultrasound," European Journal of Ultrasound, vol. 9, pp. 3-9, 1999.
[6] L. D. Johns, "Nonthermal Effects of Therapeutic Ultrasound: The
Frequency Resonance Hypothesis," Journal of Athletic Training, vol. 37,
pp. 293-299, 2002.
[7] E. L. Carstensen, "Biological effects of acoustic cavitation," Ultrasound in Medicine & Biology, vol. 12, pp. 703-704, 1986.
[8] M. A. Dinno, M. Dyson, S. R. Young, A. J. Mortimer, J. Hart, and L. A. Crum, "The significance of membrane changes in the safe and effective use of therapeutic and diagnostic ultrasound," Physics in Medicine and Biology, vol. 34, p. 1543, 1989.
[9] A. J. Mortimer and M. Dyson, "The effect of therapeutic ultrasound on calcium uptake in fibroblasts," Ultrasound Med Biol, vol. 14, pp.
499-506, 1988.
[10] F. Sachs, "Mechanical transduction in biological systems," Crit Rev
Biomed Eng, vol. 16, pp. 141-69, 1988.
[11] M. Dyson and M. Brookes, "Stimulation of bone repair by ultrasound," Ultrasound Med Biol, vol. Suppl 2, pp. 61-6, 1983.
[12] A. Ramirez, J. A. Schwane, C. McFarland, and B. Starcher, "The effect of ultrasound on collagen synthesis and fibroblast proliferation in vitro," Med Sci Sports Exerc, vol. 29, pp. 326-32, 1997.
[13] K. Ebisawa, K. Hata, K. Okada, K. Kimata, M. Ueda, S. Torii, et al.,
"Ultrasound enhances transforming growth factor beta-mediated
chondrocyte differentiation of human mesenchymal stem cells," Tissue
Eng, vol. 10, pp. 921-9, 2004.
[14] A. Katiyar, R. L. Duncan, and K. Sarkar, "Ultrasound stimulation
increases proliferation of MC3T3-E1 preosteoblast-like cells," Journal
of Therapeutic Ultrasound, vol. 2, pp. 1-1, 2014.
[15] P. G. De Deyne and M. Kirsch-Volders, "In vitro effects of therapeutic ultrasound on the nucleus of human fibroblasts," Phys Ther, vol. 75, pp. 629-34, 1995.
[16] S. R. Young and M. Dyson, "Effect of therapeutic ultrasound on the
healing of full-thickness excised skin lesions," Ultrasonics, vol. 28, pp.
175-80, 1990.
[17] S. R. Young and M. Dyson, "Macrophage responsiveness to therapeutic ultrasound," Ultrasound Med Biol, vol. 16, pp. 809-16, 1990.
[18] M. Schafer, S. Dubin, A. Geshury, and F. Ko, "Experimental methods for ultrasonically enhanced wound healing," in Ultrasonics Symposium, 1994. Proceedings., 1994 IEEE, pp. 1853-1856 vol.3, 1994.
[19] A. A. Pilla, M. Figueiredo, P. Nasser, S. Lattuga, T. Kristianseni, J.
Heckman, et al., "Non-invasive Low Intensity Ultrasound Accelerates
Bone Repair: Rabbit Fibula Model And Human Colles''and Tibial Fractures," in Engineering in Medicine and Biology Society, 1990.,
Proceedings of the Twelfth Annual International Conference of the
IEEE, pp. 1573-1574, 1990.
[20] A. R. Crisci and A. L. Ferreira, "Low-intensity pulsed ultrasound
accelerates the regeneration of the sciatic nerve after neurotomy in rats,"
Ultrasound in Medicine & Biology, vol. 28, pp. 1335-1341, 2002.
[21] 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, vol. 17, pp. 488-94, 1999.
[22] Z. Zhang, J. Huckle, C. A. Francomano, and R. G. Spencer, "The
influence of pulsed low-intensity ultrasound on matrix production of
chondrocytes at different stages of differentiation: an explant study,"
Ultrasound Med Biol, vol. 28, pp. 1547-53, 2002.
[23] Z. J. Zhang, J. Huckle, C. A. Francomano, and R. G. Spencer, "The
effects of pulsed low-intensity ultrasound on chondrocyte viability,
proliferation, gene expression and matrix production," Ultrasound Med
Biol, vol. 29, pp. 1645-51, 2003.
[24] F. Lejbkowicz, M. Zwiran, and S. Salzberg, "The response of normal
and malignant cells to ultrasound in vitro," Ultrasound Med Biol, vol. 19,
pp. 75-82, 1993.
[25] F. Lejbkowicz and S. Salzberg, "Distinct sensitivity of normal and
malignant cells to ultrasound in vitro," Environmental Health
Perspectives, vol. 105, pp. 1575-1578, 1997.
[26] S. Takeuchim, T. Watanabe, T. Sato, H. Nisimura, and N. Kawasima, "Basic study on the effect of ultrasound exposure upon suppression of cancer cell proliferation," in Ultrasonics Symposium, IEEE, pp. 1319-1322 vol.2 , 2001.
[27] J. A. S. Jerrold T. Bushberg, Edwin M. Leidholdt,John M. Boone, "The Essential Physics of Medical Imaging," vol. Third Edition, 2011.
[28] S. Wen-Yao, M. Schafer, S. Dubin, M. O''Connor, C. Ozturk, and C.
Min-Chih, "Skin temperature and impedance measurement in ultrasound
wound treatment," in Biomedical Engineering Conference, pp. 348-350,
1996.
[29] J. J. Kaufman, H. Popp, A. Chiabrera, and A. A. Pilla, "The effect of
ultrasound on the electrical impedance of biological cells," in
Engineering in Medicine and Biology Society. Proceedings of the
Annual International Conference of the IEEE, pp. 753-754 vol.2, 1988.
[30] 莊克士, "醫學影像物理學," 合記圖書, 2012.
[31] "Panametrics-NDT, Ultrasonic transducer technical notes,"
https://www.olympus-ims.com/.
[32] M. B. S. K. Kirk Shung, and B. Tsui, "Principles of Medical Imaging,"
Academic Press, 1992.
[33] D. van der Kooy and S. Weiss, "Why stem cells?," Science, vol. 287, pp. 1439-41, 2000.
[34] E. Y. Snyder and A. L. Vescovi, "The possibilities/perplexities of stem cells," Nat Biotechnol, vol. 18, pp. 827-8, 2000.
[35] N. D. Theise, S. Badve, R. Saxena, O. Henegariu, S. Sell, J. M.
Crawford, et al., "Derivation of hepatocytes from bone marrow cells in
mice after radiation-induced myeloablation," Hepatology, vol. 31, pp.
235-40, 2000.
[36] D. Orlic, J. Kajstura, S. Chimenti, I. Jakoniuk, S. M. Anderson, B. Li, et al., "Bone marrow cells regenerate infarcted myocardium," Nature, vol. 410, pp. 701-5, 2001.
[37] C. Conrad, H. Niess, R. Huss, S. Huber, I. von Luettichau, P. J. Nelson, et al., "Multipotent mesenchymal stem cells acquire a lymphendothelial phenotype and enhance lymphatic regeneration in vivo," Circulation, vol. 119, pp. 281-9, 2009.
[38] A. M. Dimarino, A. I. Caplan, and T. L. Bonfield, "Mesenchymal stem cells in tissue repair," Front Immunol, vol. 4, p. 201, 2013.
[39] A. J. Friedenstein, "Precursor cells of mechanocytes," Int Rev Cytol, vol. 47, pp. 327-59, 1976.
[40] S. E. Haynesworth, J. Goshima, V. M. Goldberg, and A. I. Caplan,
"Characterization of cells with osteogenic potential from human
marrow," Bone, 1992.
[41] M. F. Pittenger, A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mosca, et al., "Multilineage potential of adult human mesenchymal stem cells," Science, vol. 284, pp. 143-7, 1999.
[42] K. E. Hatzistergos, H. Quevedo, B. N. Oskouei, Q. Hu, G. S.
Feigenbaum, I. S. Margitich, et al., "Bone marrow mesenchymal stem
cells stimulate cardiac stem cell proliferation and differentiation," Circ
Res, vol. 107, pp. 913-22, 2010.
[43] S. Ghannam, C. Bouffi, F. Djouad, C. Jorgensen, and D. Noel,
"Immunosuppression by mesenchymal stem cells: mechanisms and
clinical applications," Stem Cell Res Ther, vol. 1, p. 2, 2010.
[44] Y. Jiang, B. N. Jahagirdar, R. L. Reinhardt, R. E. Schwartz, C. D. Keene, et al., "Pluripotency of mesenchymal stem cells derived from adult marrow," Nature, vol. 418, pp. 41-9, 2002.
[45] K. D. Lee, T. K. Kuo, J. Whang-Peng, Y. F. Chung, C. T. Lin, S. H.
Chou, et al., "In vitro hepatic differentiation of human mesenchymal
stem cells," Hepatology, vol. 40, pp. 1275-84, 2004.
[46] K. Fukuda, "Development of regenerative cardiomyocytes from
mesenchymal stem cells for cardiovascular tissue engineering," Artif
Organs, vol. 25, pp. 187-93, 2001.
[47] S. Makino, K. Fukuda, S. Miyoshi, F. Konishi, H. Kodama, J. Pan, et al., "Cardiomyocytes can be generated from marrow stromal cells in vitro," J Clin Invest, vol. 103, pp. 697-705, 1999.
[48] C. Toma, M. F. Pittenger, K. S. Cahill, B. J. Byrne, and P. D. Kessler, "Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart," Circulation, vol. 105, pp. 93-8, 2002.
[49] S. Tomita, R. K. Li, R. D. Weisel, D. A. Mickle, E. J. Kim, T. Sakai, et al., "Autologous transplantation of bone marrow cells improves
damaged heart function," Circulation, vol. 100, pp. Ii247-56, 1999.
[50] 林鉦庭, "多頻道超音波驅動系統之開發," 私立義守大學生物醫學工程學系碩士學位論文, 2011.
[51] 趙恆生, "感應充電器耦合電路設計研究," 私立中原大學電機工程學系碩士學位論文, 2001.
[52] 陳逸鴻, "低頻超音波相位陣列系統設計," 私立中原大學電機工程學系碩士學位論文, 2001.