低強度脈衝超音波刺激系統開發及其對間質幹細胞分化成心肌細胞之影響

Development of a LIPUS System on the Differentiation of Mesenchymal Stem Cells into Cardiomyocytes.

胡家豪(Jia-Hao Hu)

指導教授 ： 朱堃誠

義守大學/醫學院/生物醫學工程學系/碩士(2015年)

https://doi.org/10.6343/ISU.2015.00185

摘要

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。

關鍵字

低強度脈衝超音波 ； 間質幹細胞 ； 心肌細胞

並列摘要

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.

並列關鍵字

Low-intensity Pulsed Ultrasound ； Mesenchymal Stem Cells ； Cardiomyocytes

參考文獻

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