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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王兆麟(Jaw-Lin Wang) | |
dc.contributor.author | Hsiao-Ching Chen | en |
dc.contributor.author | 陳筱晴 | zh_TW |
dc.date.accessioned | 2021-06-17T08:41:28Z | - |
dc.date.available | 2021-08-18 | |
dc.date.copyright | 2019-08-18 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-07 | |
dc.identifier.citation | Watson, T., Therapeutic Ultrasound. 2017.
Part3 REFLECITON AND TRANSMISSION OF ULTRASOUND WAVES. Available from: http://www.fast.u-psud.fr/~martin/acoustique/support/r%C3%A9flection-r%C 3%A9fraction.pdf. O'Brien, W.D., Ultrasound-biophysics mechanisms. Progress in Biophysics & Molecular Biology, 2007. 93(1-3): p. 212-255. Fung, C.H., et al., Osteocytes exposed to far field of therapeutic ultrasound promotes osteogenic cellular activities in pre-osteoblasts through soluble factors. Ultrasonics, 2014. 54(5): p. 1358-1365. Secomski, W., et al., In vitro ultrasound experiments: Standing wave and multiple reflections influence on the outcome. Ultrasonics, 2017. 77: p. 203-213. Hensel, K., M.P. Mienkina, and G. Schmitz, ANALYSIS OF ULTRASOUND FIELDS IN CELL CULTURE WELLS FOR IN VITRO ULTRASOUND THERAPY EXPERIMENTS. Ultrasound in Medicine and Biology, 2011. 37(12): p. 2105-2115. Imashiro, C., et al., Cell Patterning Method on a Clinically Ubiquitous Culture Dish Using Acoustic Pressure Generated From Resonance Vibration of a Disk-Shaped Ultrasonic Transducer. Ieee Transactions on Biomedical Engineering, 2019. 66(1): p. 111-118. Fung, C.H., et al., Investigation of rat bone fracture healing using pulsed 1.5 MHz, 30 mW/cm(2) burst ultrasound - Axial distance dependency. Ultrasonics, 2014. 54(3): p. 850-859. Nelson, T.R., et al., Ultrasound Biosafety Considerations for the Practicing Sonographer and Sonologist. Journal of Ultrasound in Medicine, 2009. 28(2): p. 139-150. Ahmadi, F., et al., Bio-effects and safety of low-intensity, low-frequency ultrasonic exposure. Progress in Biophysics & Molecular Biology, 2012. 108(3): p. 119-138. Aliabouzar, M., L.G. Zhang, and K. Sarkar, Lipid Coated Microbubbles and Low Intensity Pulsed Ultrasound Enhance Chondrogenesis of Human Mesenchymal Stem Cells in 3D Printed Scaffolds. Scientific Reports, 2016. 6: p. 11.doi:10.6342/NTU20190276258 Doan, N., et al., In vitro effects of therapeutic ultrasound on cell proliferation, protein synthesis, and cytokine production by human fibroblasts, osteoblasts, and monocytes. Journal of Oral and Maxillofacial Surgery, 1999. 57(4): p. 409-419. Mundi, R., et al., Low-intensity pulsed ultrasound: Fracture healing. Indian Journal of Orthopaedics, 2009. 43(2): p. 132-140. Puts, R., et al., Activation of Mechanosensitive Transcription Factors in Murine C2C12 Mesenchymal Precursors by Focused Low-Intensity Pulsed Ultrasound (FLIPUS). Ieee Transactions on Ultrasonics Ferroelectrics and Frequency Control, 2016. 63(10): p. 1505-1513. Ikeda, K., et al., Effects of low-intensity pulsed ultrasound on the differentiation of C2C12 cells. Life Sciences, 2006. 79(20): p. 1936-1943. Puts., R., et al., Functional regulation of YAP mechanosensitive transcriptional coactivator by Focused Low-Intensity Pulsed Ultrasound(FLIPUS) enhances proliferation of murine mesenchymal precursors. PLOS ONE, 2018. Abrunhosa, V.M., et al., INDUCTION OF SKELETAL MUSCLE DIFFERENTIATION IN VITRO BY THERAPEUTIC ULTRASOUND. Ultrasound in Medicine and Biology, 2014. 40(3): p. 504-512. Salgarella, A.R., et al., OPTIMAL ULTRASOUND EXPOSURE CONDITIONS FOR MAXIMIZING C2C12 MUSCLE CELL PROLIFERATION AND DIFFERENTIATION. Ultrasound in Medicine and Biology, 2017. 43(7): p. 1452-1465. Johns, L.D., Nonthermal effects of therapeutic ultrasound: The frequency resonance hypothesis. Journal of Athletic Training, 2002. 37(3): p. 293-299. Wu, J.R., Shear stress in cells generated by ultrasound. Progress in Biophysics & Molecular Biology, 2007. 93(1-3): p. 363-373. Watson, T., The role of electrotherapy in contemporary physiotherapy practice. Manual Therapy, 2000. 5(3): p. 132-141. Rawool, N.M., et al., Power Doppler assessment of vascular changes during fracture treatment with low-intensity ultrasound. Journal of Ultrasound in Medicine, 2003. 22(2): p. 145-153. Karshafian, R., et al., SONOPORATION BY ULTRASOUND-ACTIVATED MICROBUBBLE CONTRAST AGENTS: EFFECT OF ACOUSTIC EXPOSURE PARAMETERS ON CELL MEMBRANE PERMEABILITY AND CELL VIABILITY. Ultrasound in Medicine and Biology, 2009. 35(5): p. 847-860. Leskinen, J.J. and K. Hynynen, STUDY OF FACTORS AFFECTING THEMAGNITUDE AND NATURE OF ULTRASOUND EXPOSURE WITH IN VITRO SET-UPS. Ultrasound in Medicine and Biology, 2012. 38(5): p. 777-794. Puts, R., et al., A Focused Low-Intensity Pulsed Ultrasound (FLIPUS) System for Cell Stimulation: Physical and Biological Proof of Principle. Ieee Transactions on Ultrasonics Ferroelectrics and Frequency Control, 2016. 63(1): p. 91-100. Musar, #xf2, and A. , The Basis of Muscle Regeneration. Advances in Biology, 2014. 2014: p. 16. Goetsch, K.P., K.H. Myburgh, and C.U. Niesler, In vitro myoblast motility models: investigating migration dynamics for the study of skeletal muscle repair. Journal of Muscle Research and Cell Motility, 2013. 34(5-6): p. 333-347. Burattini, S., et al., C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. European Journal of Histochemistry, 2004. 48(3): p. 223-233. McMahon, D.K., et al., C2C12 CELLS - BIOPHYSICAL, BIOCHEMICAL, AND IMMUNOCYTOCHEMICAL PROPERTIES. American Journal of Physiology, 1994. 266(6): p. C1795-C1802. Engler, A.J., et al., Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments. Journal of Cell Biology, 2004. 166(6): p. 877-887. Kramerova, I., E. Kudryashova, and M.J. Spencer, Null mutation of calpain 3(p94) in mice causes abnormal sarcomere formation in vivo and in vitro. Human Molecular Genetics, 2004. 13(13): p. 1373-1388. Meng, Z.P., T. Moroishi, and K.L. Guan, Mechanisms of Hippo pathway regulation. Genes & Development, 2016. 30(1): p. 1-17. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74538 | - |
dc.description.abstract | 超音波在臨床上已經被證實能有效地加速骨癒合,因此許多學者開始探討超音波對不同種類細胞,例如肌肉細胞,是否會有類似的功能。本實驗的前導實驗使用文獻中常見的超音波參數來刺激細胞,實驗結果發現此能量似乎過大,細胞無法貼覆在培養皿,此外使用超音波探頭直接施打到培養皿亦會有能量不平均的現象。因此,本實驗設計一創新設計,可將能量均勻地分布到培養皿上。我們使用一均勻分布之微能量(0.02~0.84mW/cm 2 )超音波刺激 C2C12 肌母細胞,探討此一超音波能量是否對 C2C12 肌母細胞遷移(Migration)、增生Proliferation)以及分化(Differentiation)有所影響。
在微能量超音波刺激後,C2C12 肌母細胞的平均遷移速度加快 1.2 倍,傷口癒合實驗的面積恢復速度則加快 1.3 倍。在增生實驗中,細胞在連續刺激三天超音波並且隔一天反應後,對於控制組的細胞數量有顯著增加(p=0.02)。 在分化實驗中,超音波刺激後形成的肌管,其平均長度為控制組肌管的 1.4倍,平均寬度則為控制組的 1.1 倍。透過螢光染色方法來分析結果,微能量超音波促使肌管擁有更多的核融合比例(Fusion Index),同時肌管也含有較廣分布和較高表現量的 Slow Skeletal Myosin Heavy Chain。在肌原纖維的觀察中,刺激後形成的肌管,粗略計算含有更多的纖維數量,此現象經他人重複實驗可得到相同結果。在肌節的部分,兩組皆有觀察到肌節,但因為目前所能判斷的數據不夠,因此無法明確的下判定。 本實驗使用的微能量超音波在完全不傷害細胞的狀況下,仍具有加速C2C12肌母細胞遷移、增生以及分化的能力。至於微能量超音波對肌母細胞分化後功能性的影響,仍須進一步的探討。 | zh_TW |
dc.description.abstract | The ultrasound has been successfully used for bone healing. Many researchers hence to explore the possibility of using ultrasound for different type of cells, e.g. the muscle cell. In the pilot study of this research, we used the convention parameters for bone stimulations to test the feasibility for muscle growth. We found the cell cannot adhere to the patch dish due to the excitation energy. In addition, the distribution of applied energy was not evenly distributed onto the patch dish. Therefore, we designed an instrument that can evenly distributed the energy among the incubation area. This instrument can apply an evenly distributed low intensity energy (0.02~0.84mW/cm 2 ) to stimulate the C2C12 cells, and find correspondence consequence of ultrasound stimulations on the cell migration, proliferation, and differentiation.
The results showed that the migration of C2C12 is 1.2 times faster after stimulation, while the rate of wound recover is 1.3 times faster than control. In respect to proliferation, the cell number was significantly higher (p=0.02) than control after three-days of continuous treatment. In respect to differentiation, the width of myotube was 1.1 times wider than the control; while the length of myotube was 1.4 times longer than the control after ultrasonic stimulation. Through inflorescence analysis, myotube generation under ultrasound stimulation had a higher fusion index as well as distribution and expression of slow skeletal myosin heavy chain. In addition, more myofibril was observed after a brief analysis. This phenomenon was observed again in the repeated experiment by a colleague. Although there were some differences observed in the morphology of sarcomere between the control and treated group, there was not enough data to make a conclusion. This study showed that the non-thermal and very low intensity ultrasound does accelerate the regeneration process of C2C12 cell without any damage to cell. The ultrasonic effect on myotube function is worth of further investigation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:41:28Z (GMT). No. of bitstreams: 1 ntu-108-R06548035-1.pdf: 3821047 bytes, checksum: 6e6b87e29d1e86f098c0cf4445216924 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 致謝 ........................................................................................................................ I
中文摘要....................................................................................................................... II Abstract ...................................................................................................................... III 目錄 ....................................................................................................................... V 圖表目錄...................................................................................................................... IX 第一章 緒論................................................................................................................ 1 1.1 醫用超音波.................................................................................................... 1 1.1.1 超音波定義及簡介.............................................................................. 1 1.1.2 空間能量分佈...................................................................................... 1 1.1.3 脈衝重複頻率(PRF)、涵蓋率及強度 ................................................ 2 1.1.4 醫用超音波應用.................................................................................. 2 1.2 超音波刺激細胞............................................................................................. 3 1.2.1 細胞反應可能的機制.......................................................................... 3 1.2.2 超音波刺激細胞系統.......................................................................... 4 1.3 肌肉再生......................................................................................................... 5 1.3.1 衛星細胞(Satellite cells)轉換成肌管 ................................................. 6 1.3.2 肌肉再生研究 MODEL: C2C12 ......................................................... 6 1.4 超音波刺激 C2C12 肌母細胞文獻回顧 ....................................................... 7 1.5 前導實驗......................................................................................................... 8 1.6 實驗目的......................................................................................................... 9 第二章 材料與方法.................................................................................................. 10 2.1 研究方法介紹............................................................................................... 10 2.2 超音波刺激系統........................................................................................... 10 2.2.1 超音波訊號輸出端............................................................................ 11 2.2.2 超音波刺激細胞端............................................................................ 12 2.3 使用設備....................................................................................................... 13 2.3.1 水聽器(Hydrophone) ......................................................................... 13 2.3.2 電漿清洗機(Plasma cleaner) ............................................................. 14 2.3.3 細胞計數盤(Hemocytometer) ........................................................... 14 2.4 超音波強度和聲壓量測方法....................................................................... 15 2.4.1 強度和聲壓換算................................................................................ 16 2.5 超音波載台內升溫量測............................................................................... 17 2.6. C2C12 肌母細胞實驗 .................................................................................. 18 2.6.1 C2C12 肌母細胞培養 ........................................................................ 18 2.6.2 C2C12 肌母細胞遷移(Migration)實驗.............................................. 18 2.6.3 C2C12 肌母細胞增生(Proliferation)和活性(Viability)實驗 ............. 19 2.6.4 C2C12 肌母細胞分化(Differentiation)實驗 ...................................... 20 2.6.4.1 免疫螢光染色(Immunofluorescence ) ................................... 21 2.6.4.2 西方墨點轉漬法(Western blot) .............................................. 22 2.7 軟體分析........................................................................................................ 26 2.7.1 C2C12 肌母細胞遷移(Migration)...................................................... 26 2.7.2 C2C12 肌母細胞增生(Proliferation)和活性(Viability) ..................... 27 2.7.3 C2C12 肌母細胞分化(Differentiation) .............................................. 27 2.7.3.1 Bright field 影像之肌管長寬分析 .......................................... 27 2.7.3.2 螢光染色之 Slow Skeletal Myosin Heavy Chain 面積比例 . 27 2.7.3.3 螢光染色之核融合比例(Fusion Index) ................................. 28 2.7.3.4 螢光強度計算方法................................................................. 29 2.7.3.5 肌原纖維數量......................................................................... 30 2.7.3.6 西方墨點轉漬法(Western blot) .............................................. 30 第三章 實驗結果...................................................................................................... 31 3.1 超音波系統................................................................................................... 31 3.1.1 超音波強度和聲壓量測.................................................................... 31 3.1.2 升溫量測............................................................................................ 32 3.2 C2C12 肌母細胞遷移(Migration)實驗......................................................... 32 3.3 C2C12 肌母細胞增生(Proliferation)實驗 .................................................... 34 3.4 C2C12 肌母細胞活性(Viability)實驗 ........................................................... 34 3.5 C2C12 肌母細胞分化(Differentiation) ......................................................... 35 3.5.1 種植細胞濃度和 Slow Skeletal Myosin Heavy Chain 蛋白測試 .... 35 3.5.2 肌管長寬比較.................................................................................... 36 3.5.3 Slow Skeletal Myosin Heavy Chain 面積比例 .................................. 38 3.5.4 核總數................................................................................................ 40 3.5.5 核融合比例(Fusion index) ................................................................ 41 3.5.6 平均 Slow Muscle Myosin Heavy Chain 螢光強度分析 ................. 42 3.5.7 肌原纖維(Myofibril)觀察 ................................................................. 45 3.5.8 肌節(Z-body / Sarcomere)觀察 ......................................................... 46 第四章 討論.............................................................................................................. 47 4.1 超音波系統................................................................................................... 47 4.2 C2C12 肌母細胞實驗 ................................................................................... 48 4.2.1 不同批 C2C12 肌母細胞特性 .......................................................... 48 4.2.2 C2C12 肌母細胞遷移 ........................................................................ 48 4.2.3 C2C12 肌母細胞增生及活性 ............................................................ 49 4.2.4 超音波促進 C2C12 分化之文獻討論 .............................................. 50 4.2.5 肌管型態............................................................................................ 51 4.2.6 肌管 Slow Skeletal Myosin Heavy Chain 表現量和核數 ................ 52 4.2.7 肌原纖維和肌節的觀察.................................................................... 53 4.2.8 C2C12 肌母細胞分化西方墨點轉漬法 ............................................ 54 4.3 實驗限制....................................................................................................... 55 4.3.1 超音波系統限制................................................................................ 55 4.3.2 部分分析判斷標準及實驗手法........................................................ 55 4.3.3 C2C12 肌母細胞培養 ........................................................................ 55 第五章 結論與未來展望.......................................................................................... 56 5.1 結論............................................................................................................... 56 5.2 未來展望....................................................................................................... 56 參考文獻...................................................................................................................... 57 | |
dc.language.iso | zh-TW | |
dc.title | 微能量超音波對肌母細胞 C2C12遷移、增生以及分化的影響 | zh_TW |
dc.title | Effect of Very Low Intensity Ultrasound (VLIUS) on C2C12 Myoblast Migration, Proliferation and Differentiation | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張孜菁,林文澧(Win-Li Lin) | |
dc.subject.keyword | C2C12 肌母細胞,微能量超音波,肌肉再生,遷移,增生,分化, | zh_TW |
dc.subject.keyword | C2C12 myoblast,Very-low-intensity ultrasound,Muscle regeneration,Migration,Proliferation,Differentiation, | en |
dc.relation.page | 59 | |
dc.identifier.doi | 10.6342/NTU201902762 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-07 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
顯示於系所單位: | 醫學工程學研究所 |
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