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標題: | 微能量超音波對肌母細胞 C2C12遷移、增生以及分化的影響 Effect of Very Low Intensity Ultrasound (VLIUS) on C2C12 Myoblast Migration, Proliferation and Differentiation |
作者: | Hsiao-Ching Chen 陳筱晴 |
指導教授: | 王兆麟(Jaw-Lin Wang) |
關鍵字: | C2C12 肌母細胞,微能量超音波,肌肉再生,遷移,增生,分化, C2C12 myoblast,Very-low-intensity ultrasound,Muscle regeneration,Migration,Proliferation,Differentiation, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74538 |
DOI: | 10.6342/NTU201902762 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學工程學研究所 |
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