- 學位論文
超音波微管在細胞上的應用
Development of Micropipette Ultrasound for Cellular Stimulation
摘要
實驗背景： 超音波刺激已經廣泛應用在生物組織以及細胞上，各個實驗室設計自己的超音波刺激工具並利用不同的蛋白質標定物來分析細胞在受到超音波刺激時所產生的生物反應，並提出極具貢獻的研究成果。至今不少研究提出細胞在受超音波刺激時會促進增生速率以及在組織上甚至能開啟血腦屏障，然而鮮少研究提及超音波上不同的物理效應在生物實驗上主導的反應，並且對於不同的超音波工具會因為工具的尺寸，幾何以及超音波傳遞方式而出現諸多限制使得一種工具無法應付多樣性的生物實驗。 設計及開發工具的動機： 本研究開發的超音波微管將玻璃管當作波導(Waveguide)傳遞超音波至細胞液體以達到刺激細胞的目的，本實驗室過去開發的超音波微管無法穩定的在「超音波微管影響細胞外觀變化」以及「超音波開啟細胞離子通道」的生物實驗上得到穩定的結果，原因是過去的超音波微管所能輸出的能量不足導致。 工具的設計及開發： 基於過去超音波微管無法傳遞足夠的能量到欲刺激的細胞，本研究改良了微管的幾何以及減少超音波傳遞時經由不同介質的邊界數量來提高超音波的輸出能量。在工具設計與開發的章節中，本研究提供經過校正的水聽器(Hydrophone)並敘述校正方法以提高超音波微管輸出的可信度也比較了過去開發的超音波微管以及本研究開發的超音波微管對於超音波能量傳遞的差異。 理論上，超音波微細管末端的管徑小於超音波的波長因此可將其視為一個在液體中的點波源。本研究利用物理實驗測量微管垂直以及水平偏移時超音波能量的衰減驗證超音波微管是局部性的刺激工具。最後利用高倍顯微鏡的即時攝影發現超音波微管會對流體造成速度場，過去的研究也提到超音波探頭在液體中會產生兩種物理效應：超音波的聲輻射以及超音波造成的液體流動現象，我們將此速度場作分析並得到與微管越遠的區域的速度比離微管較近的區域之速度小，也討論電源輸入到微管的電壓峰對峰值決定了超音波微管輸出壓力(超音波輻射壓力)以及強度(超音波造成流體運動)的大小，而佔空比(Duty factor)則影響了強度(超音波造成流體運動)。 超音波微管在細胞上的局部性刺激： 本章節建立在超音波微管是局部性超音波刺激工具的基礎下進行的生物實驗，在微管輸出的超音波能量提升的基礎下施行超音波微管影響細胞外觀變化的生物實驗，並觀察到細胞骨架在超音波刺激時局部性的變化。在觀察到細胞骨架在超音波刺激時局部性的變化之後我們藉由神經細胞鈣離子濃度變化的生物實驗得出距離超音波微管較遠的細胞在超音波作用之下鈣離子濃度螢光的亮度(灰值)比較近的細胞低，驗證超音波微管是局部性的刺激工具，鑒於超音波微管在液體中產生兩種物理效應：超音波的聲輻射以及超音波造成的液體流動現象，以及局部性的刺激，我們也假設了超音波的液體流動會影響神經細胞的 GPCRs，在神經細胞內加入 G protein 的拮抗劑來抑制並神經細胞內鈣離子濃度的增加，不過結果顯示細胞內的鈣離子在超音作用時仍有上升，並且隨著微管與細胞的距離增加神經細胞鈣離子濃度的減少的幅度在加入 G protein 時並無改變，顯示超音波造成的液體流動並非影響神經細胞 GPCRs 的主因，聲輻射以及超音波造成的液體流動皆有扮演刺激細胞的角色。 超音波微管複合力在細胞上的應用： 驗證了超音波對於神經細胞有兩種物理刺激：超音波聲輻射以及超音波作用時液體中產生流動現象的特性(複合外力)對於髓核細胞(Nucleus pulposus)以及小鼠肌母細胞(C2C12)受到超音波微管的複合力刺激時胞內鈣離子濃度分析，兩種物理效應在細胞刺激上的權重。 得知超音波聲輻射以及超音波作用時液體中產生流動現象在細胞刺激上的地位之後，我們更深入探討了這些效應在細胞上的應用。超音波微管在老鼠背神經節細胞(DRG Neuron cell) 經歷超音波微管施予超音波之後細胞膜上膜電流(電位)的變化產生了向內電流(Inward current)，最後利用兩種離子通道阻斷劑(Amiloride 以及 APETx2)尋找細胞在受超音波刺激時開啟的離子通道為 ASIC3。 結論： 超音波微管的輸出能量有顯著的提升是在生物實驗中穩定的超音波刺激工具，並且其幾何的形狀以及尺寸的大小可以適應多樣化的實驗環境。
並列摘要
Introduction: Ultrasonic stimulation has been widely used in biological tissues and cells. Each laboratory designs its own ultrasonic stimulation tools and uses different protein markers to analyze the biological response of cells stimulated by ultrasonic waves having made great contributions. So far, many studies have proposed that cells can promote the proliferation rate and even open the blood-brain barrier on tissues during ultrasound stimulation. However, few studies mentioned the different physical effects generated by ultrasound influencing the biological experiments. Furthermore, ultrasonic tools will have many limitations due to the tool's size, geometry, and ultrasonic transmission methods, making one tool unable to cope with diverse biological experiments. Motivation of Tool development: The acoustic micropipette developed in this research uses a glass tube as a waveguide to transmit ultrasonic waves to stimulate the cells in media. The prototype of acoustic micropipette developed in the previous study of our laboratory was not able to work stably in the biological experiments of “Acoustic micropipette affects the morphological characteristics of cells” and “Ion channel opening during ultrasound stimulation” because of insufficient energy delivered. Thus, we need to improve output power of this tool. Design and development: As the previous acoustic micropipette could not deliver enough energy to the cells, this study improved the geometry of the acoustic micropipette and reduced the number of boundaries between different materials during ultrasonic transmission to increase the ultrasonic output energy. In the chapter of tool design and development, this research describes the hydrophone calibration method measuring output of acoustic micropipette and then provides output performance of this tool. Theoretically, the diameter of the end of the acoustic micropipette is smaller than the wavelength of the ultrasonic wave, so it can be regarded as a point source in the liquid. We utilized physical experiments to measure the attenuation of ultrasonic energy when micropipette is displaced vertically and horizontally to verify that acoustic micropipette is a kind of local stimulation tool. Finally, real-time photography using a high-speed microscope found that acoustic micropipette would create acoustic streaming in the media. Studies in the past also mentioned that ultrasonic probes will produce two physical effects in liquids: acoustic radiation and fluid flow caused by ultrasonic waves. We analyzed this velocity field and found that the velocity of the area farther from the micropipette is lower than the velocity of the area closer to the micropipette. Besides, we also discussed that peak-to-peak voltage of the power input to the acoustic micropipette determines output pressure level (acoustic radiation pressure) and intensity (ultrasonic waves cause fluid movement) but duty factor affects only intensity (ultrasonic waves cause fluid movement). Local cell stimulation of acoustic micropipette: This chapter, based on acoustic micropipette being a local ultrasonic stimulation tool, exhibits some biological experiments by acoustic micropipette in new design. Starting off with “Acoustic micropipette affects the morphological characteristics of cells”, we observed the local changes of the cytoskeleton under ultrasound stimulation. Depending on local changes of the cytoskeleton during ultrasound stimulation, we detected the concentration of intracellular calcium in neuron cells and then discovered that calcium ion concentration of cells farther from the micropipette is lower than closer during ultrasound stimulation. Otherwise, we also hypothesized that the fluid flow of ultrasound will affect the GPCRs (G protein-coupled receptors) because GPCRs play pivotal roles in regulating the function and plasticity of neuronal circuits in the nervous system. Thus, this study also applied G protein antagonist in neuron cell then repeating the calcium image experiment and the result is that liquid flow caused by ultrasound is not the only cause that affects the GPCR of nerve cells. Acoustic radiation and acoustic streaming caused by ultrasonic waves both play a role in stimulating cells. The biological effect of complex loading by acoustic micropipette: It has been verified that ultrasonic waves have two physical stimuli to cells: acoustic radiation and acoustic streaming caused by ultrasonic waves. The study utilized the unique loading to stimulate nucleus pulposus cells and C2C12 cells and observed calcium ion concentration of cells to determine acoustic radiation and fluid flow which is more important in cell stimulation. After knowing the role of ultrasonic sound radiation and acoustic streaming during ultrasonic stimulation, we would like to expand on this idea by applying these effects to DRG neuron cell electrophysiologic testing. In this testing, when the DRG neuron cell was stimulated by ultrasound from micropipette, inward current occurred due to change of the cell membrane current. In the end, we added two kinds of ion channels blockers (Amiloride and APETx2) to inhibit inward current during ultrasound. According to the results of the ion channel test for DRG neuron cells by ultrasound, APETx2 is effective in blocking inward current but Amiloride is not. Conclusion: After output energy level is enhanced, acoustic micropipette is a stable ultrasonic stimulation tool in biological experiments. In addition, its geometric shape and size can adapt to diverse experimental environments.