臺灣博碩士論文加值系統

在新藥開發的過程中，第一階段的藥物篩檢步驟佔有相當重要的角色。且所有藥物在上市前皆必須通過心臟毒性的檢驗。而在心臟藥物的開發方面，心肌細胞的收縮表現、跳動頻率與其施力曲線是判斷藥物是否有效的重要的特徵指標。因此，用於藥物篩檢之監測系統是否能監控心肌細胞的收縮及舒張力、頻率與收縮特性曲線，成為了最必要的課題。為了量化心肌的施力曲線，目前的監測系統大多以光學系統配合高解析度相機為主要觀測方法，監控測細胞肌節的收縮或柔性基材的形變量，然後以影像分析與數值運算，估算形變量，並轉換為應力。然而，此一方法有數個重大限制：第一，使用基材變形而造成的影像形變量來推算力為其間接訊號，所推算出的應力並不能完全代表心肌真正的收縮力；第二，影像處理與運算系統需要專業人員操作，難以達到全自動化的目標。第三，為維持足夠之解析度而受限於光學系統的視野，，使得能監測之裝置數量有限，無法同時進行大量地檢測。
為了排除光學系統之根本限制，同時為了達成自動化、即時性的、及量測施力曲線之能力，本研究開發一可應用於心肌–壓電耦合系統之心臟藥物篩檢系統。心肌-壓電耦合平台是以聚偏二氟乙烯柔性薄膜作為基材，透過心肌細胞收縮帶動基材形變輸出電訊號之心肌訊號量測平台。而心臟藥物篩檢系統則是以此平台為基礎，對平台輸出之訊號進行外部雜訊屏蔽、促進平台心肌細胞成長以及測量平台形變之電訊號與心肌細胞施力之關係。最後，此心臟藥物篩檢系統以兩種商業化之心臟藥物進行測試，分別為異丙腎上腺素與美托洛爾β₁-受體阻斷劑。實驗結果證實此一心肌–壓電耦合系統可成功進行心肌細胞施力曲線之即時性量測，並且能夠量測到在藥物作用下，心肌施力之變化。
使用異丙腎上腺素進行藥物試驗之結果可使心肌細胞頻率與施力顯著上升，並且使用商用軟體計算所得之EC50 為200 nM 符合過去文獻提及之數值誤差範圍內，使用美托洛爾同樣可發現心肌細胞之頻率與施力在下降，且IC50 為368 μM符合過去文獻提及之數值誤差範圍內，另一方面，於研究中亦發現異丙腎上腺素在超過EC50 的62 nM 濃度，可刺激心肌細胞提升45%的施力強度，證明一般調控心律的異丙腎上腺素亦有調控施力強度的功能，驗證以壓電薄膜為感測器可同時量測心肌的跳動頻率及施力強度之功能。由此可驗證此系統之現自動化、直接性檢測心肌細胞施力訊號的可行性，達到進行可大量進行初步藥物篩檢的計畫目標。

The first step of drug development is drug screening. It is a very time consuming and is a very expensive process. In the development of cardiac drug, the concentration and frequency of cardiac tissue are important indicators to determine whether the compound is effective. To quantify concentration curve and frequency of cardiac tissue, most of monitor systems are base on optical system to monitor the deformation of substrate, including high-resolution cameras and laser displacement meter.Image analysis software are used to estimate the strain and stress of cardiac tissue. However, using image analyzing processhas some limitations: First, the analysis is based on substrate deformation, its force porfile is an indirect estimation. Second, the optical system need to be processed by experienced specialists, and it is hard to become an automatic process. Finally, it cannot perform laege number of detection at the same time.
To achieve an automatic, real-time and direct measurement of cardiac force profile, we developed a cardiac–drug screening system. The system is based on Cardiac-and-piezoelectric hybrid platform based on a polyvinylidene fluoride (PVDF) flexible film as a substrate to monitor the concentraction of cardiomyocytes. The cardiac-drug screening system shields the environmental noise from the environment, microgrooves and extracellular matrix are used to promote the growth of cardiomyocytes, and cardiac contracting behaviors are measured through the electrical signals generated by the deformation induced by cardiomyocytes. Finally, the cardiac-drug screen system was tested with two commercial drugs, isoproterenol and Metoprolol. The results of the drug test using isoproterenol resulted in a significant increase in cardiomyocyte frequency and amplitude, and the EC50 calculated using commercial software was 200 nM, which was within the numerical error range mentioned in the reference. Metoprolol was also used. It was found that the frequency and amplitude of cardiomyocytes were decreasing, and the IC50 of 368 μM was within the numerical error range mentioned in the refernce. On the other hand, isoproterenol was found to exceed the EC50 concentration of 62 nM in the study. Cardiomyocytes increased the exertion intensity by 45%, which proved that isoproterenol also has the function of regulating the contracting strength. It is verified that the piezoelectric membrane can be used as a sensor to simultaneously measure the beating frequency and the amplitude. This verifies the feasibility of the automatic and direct detection of cardiomyocyte contracting signals in this system, and we successfully achieve the goal of performing a large number of preliminary drug screening tests.

致謝…………………………………………………………………….…i
摘要………………………………………………………………………ii
Abstract………………………………………………………………….iii
目錄……………………………………………………………………...iv
圖目錄………………………………………………………………….viii
表目錄…………………………………………………………………..xv
第1章 緒論……………………………………………………………1
1.1 前言…………………………………………………………...1
1.2 研究目的……………………………………………………...2
1.3 論文架構……………………………………………………...3
第2章 文獻回顧………………………………………………………5
2.1 心肌細胞之機械與生理特性…………………………………5
2.2 促使心肌細胞成熟之方法……………………………………6
2.2.1 機械拉伸……………………………………………......7
2.2.2 電生理刺激…………………………………………......8
2.3 促進心肌細胞排列之方法…………………………………...14
2.3.1 基材幾何結構………………………………………....15
2.3.2 細胞外基質..……………………………………….….15
2.4 心肌組織的監測方法………………………………………..17
2.4.1 光學系統量測…………………………………………17
2.4.2 3D 列印系統…………………………………………..20
2.4.3 體外聚偏二氟乙烯生物換能系統……………………20
2.5 心臟藥物檢測………………………………………………..21
第3章 設計理念……………………………………………………..24
第4章 實驗方法……………………………………………………..33
4.1 心肌-壓電耦合平台………………………………………….33
4.1.1 設計理念……………………………………................33
4.1.2 低溫聚偏二氟乙烯微結構製程………...…………….36
4.1.3 心肌-壓電耦合平台改良……………………………...42
4.1.4 心肌-壓電耦合平台靈敏度測試…………………...…45
4.2 訊號監測系統………………………………………………..47
4.3 熱源供應系統………………………………………………..50
4.4 法拉第雜訊屏蔽盒…………………………………………..51
4.4.1 設計理念……………………………………………....51
4.4.2 法拉第雜訊屏蔽盒設計…………………………..…..52
4.5 電生理刺激系統……………………………………………..57
4.5.1 電刺激參數………………………………………...….58
4.5.2 電極設計………………………..……….…………….59
4.6 細胞培養……………………………………………………...61
4.6.1 心肌細胞培養液………………………………………61
4.6.2 心肌細胞解凍…………………………………………62
4.6.3 細胞外基質……………………………………………63
4.6.4 心肌細胞培養…………………………………………63
4.7 細胞染色…………………………………………………….63
4.7.1 螢光染色步驟…………………………………………65
4.7.2 光學成像系統…………………………………………66
4.8 實驗架設…………………………………………………….67
4.8.1 心肌-壓電耦合平台之訊號量測……………………...67
4.8.2 外部電生理刺激系統…………………………………67
第5章 實驗結果與討論……………………………………………..69
5.1 心肌-壓電耦合平台靈敏度測試結果………………………69
5.2 心肌細胞之電刺激與細胞染色結果……………………….70
5.2.1 外部電刺激對心肌細胞之影響……………………...70
5.2.2 心肌細胞染色結果…………………………………...71
5.3 心肌-壓電耦合平台之藥物測試……………………………78
5.3.1 實驗設計與參數說明...………………………………78
5.3.2 細胞外基質對藥物試驗影響之比較……...…………80
5.3.2.1 Isoproterenol 藥物試驗結果……………….80
5.3.2.2 Metoprolol 藥物試驗結果………………....88
5.3.3 電刺激對心肌細胞藥物試驗之影響…………….94
5.3.3.1 Isoproterenol 藥物試驗結果…………….....94
5.3.3.2 Metoprolol 藥物試驗結果……………….....98
5.3.4 HUVEC 共培養對藥物試驗影響之比較……………102
5.3.4.1 Isoproterenol 藥物試驗結果…………………..102
5.3.4.2 Metoprolol 藥物試驗結果…………………….106
第6章 結語與未來展望……………………………………………111
6.1 結語…………………………………………………………111
6.2 未來展望……………………………………………………112
參考文獻………………………………………………………………113
附錄……………………………………………………………………118
附錄A、Syringe Pump 往復運動程式………………………….118
附錄B、心肌-壓電耦合系統儀器………………………………119
附錄C、MatLab 心肌-壓電耦合系統頻率與施力分析……….120

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