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研究生: 出雅如
Chu, Ya-Ju
論文名稱: Lobule-Mimetic Drug Testing Lab Chip for the studies of Klebsiella pneumoniae-Induced Pyogenic Liver Abscess
仿肝小葉藥物檢測實驗室晶片於克雷白氏肺炎桿菌誘發化膿性肝膿瘍之研究
指導教授: 劉承賢
Liu, Cheng-Hsien
口試委員: 盧向成
Lu, Shiang-Cheng
徐琅
Hsu, Long
學位類別: 碩士
Master
系所名稱: 工學院 - 動力機械工程學系
Department of Power Mechanical Engineering
論文出版年: 2011
畢業學年度: 99
語文別: 英文
論文頁數: 76
中文關鍵詞: 化膿性肝膿瘍濃度梯度聲波混合器細胞排列
外文關鍵詞: pyogenic liver abscess, gradient, acoustic micromixer, cell patterning
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  • With a steady rise in pharmaceutical costs,personalized healthcare is gaining interest and medical care is becoming as individualized as possible. The microfluidic chips have emerged as a suitable platform to conduct in vitro drug testing to perform more efficient in vitro drug testing, building and mimicking a tissue or organ in vitro it could be more close to real human body. Pyogenic liver abscess is a common cause of the liver disease in Taiwan with diabetes as the major predisposing factor and Klebsiella pneumoniae as the primary pathogen. It is important to establish an in vitro pyogenic liver abscess model for studying this disease.

    This research presents an integrated drug testing lab chip which integrates a lobule mimetic dielectrophoretic (DEP) cell pattern to establish the pyogenic liver abscess model and a continuous concentration gradient generator with acoustic micromixer for monitoring the IL-8 secretion. DEP forces are based on polarity difference to manipulate heterogeneous cells for mimicking the inherent hepatic morphology and increasing the cell-cell interaction. The gradient generator allows simultaneous administration of drug dose at different concentration and acoustic micromixer that the vibrating bubble membrane increases the mixing efficiency which helps us to monitor the in vitro antioxidant response. It used N-Acetyl-L-Cysteine (NAC) as a drug model to assess the inflammation in a dose-dependent manner. The infected hepatoma cells induced IL-8 secretion was inhibited by the addition of NAC. Compared to conventional static culture method the proposed model provides a useful platform for in vitro drug testing.


    隨著製藥的成本大幅上升,個人化醫療檢驗及照護已逐漸成為未來之趨勢。隨著微機電產業的蓬勃發展,為了有效地在體外進行藥物檢測,微流體晶片很適合作為一藥物檢測平台將藥物以連續和具整合性的方式大量檢測,且若能在體外建立一個仿人體組織或器官排列之模型,更能接近人體的真實情況也易直接觀察。 在台灣,由克雷白氏肺炎桿菌所引起的化膿性肝膿瘍相當常見,尤其是糖尿病患者。因此,在體外建立一個化膿性肝膿瘍模型是很重要的,以用來進行研究及檢測藥物。

    本研究整合一藥物檢測實驗室晶片,運用介電泳將細胞依肝臟組織的形狀排列並用克雷白氏肺炎桿菌刺激以誘發化膿性肝膿瘍而產生發炎反應,即建立一體外化膿性肝膿瘍模型。此外,實驗室晶片具有提供連續性濃度梯度的功能,當藥物呈不同濃度分布後會透過聲波振動混合器將其混合更均勻。因此,當施予適當頻率的交流電壓時會產生介電泳效應,其依照細胞表面被極化的程度不同來操控細胞,以模仿肝組織之形態及增加細胞和細胞之間的交互作用,當肝細胞受克雷白氏肺炎桿菌感染後,會產生發炎反應為一仿生之化膿性肝膿瘍。複雜的微流道結構能夠提供一濃度梯度及聲波震動混合器藉由振動流道中的氣泡以增加混合之效果,再將不同劑量的抗氧化藥物灌流至化膿性肝膿瘍模型之中以進行觀察其介白素(Interleukin-8, IL-8)之分泌情形。實驗中,以N-乙醯基半胱氨酸(N-Acetyl-L-Cysteine , NAC)一種抗氧化劑,來驗證被克雷白氏肺炎桿菌感染後的肝細胞其分泌的介白素量的濃度梯度分布,以驗證此系統的可行性。因此,在生物醫藥應用方面,此仿肝小葉藥物檢測實驗式晶片為一個很好的藥物及診斷工具。

    Table of contents Abstract I 摘要 II 致謝 III Table of contents IV List of figures VIII Chapter 1- Introduction - 1 - 1.1 Lab-on-a-chip (LOC) - 1 - 1.2 Tissue engineering - 2 - 1.3 Microfluidic system of cell culture - 5 - 1.4 Gradient - 8 - 1.5 Acoustic micromixer - 11 - 1.6 Cell patterning technology - 15 - 1.6.1 Cell patterning by using dielectrophoresis - 15 - 1.6.2 Cell patterning using negative dielectrophoresis- 16 - 1.7 Klebsiella pneumoniae-induced liver abscess - 18 - 1.8 Antioxidants on IL-8 production - 21 - Chapter 2- Development - 23 - 2.1 Theoretical reviews - 23 - 2.1.1 Dielectrophoresis - 23 - 2.1.2 Transmembrane potential - 26 - 2.1.3 Mass transport in microscale - 27 - 2.2 Design concept - 29 - 2.2.1 Design of microfluidic system generating gradient -29- 2.2.2 Design of lobule-mimetic-stellate electrode -31 - 2.2.3 Design of acoustic micromixer - 33 - 2.3 Numerical simulation - 35 - 2.3.1 Numerical simulation of gradient - 35 - 2.3.2 Numerical simulation of lobule-mimetic-stellate electrode - 37 - 2.3.3 Numerical simulation of acoustic micromixer -39 - Chapter 3- Microfabrication - 42 - 3.1 The process of microfabrication - 42 - 3.2 The results of microfabrication - 45 - Chapter 4-Materials and Methods - 47 - 4.1 The experiments of gradient generation - 47 - 4.2 The experiments of acoustic micromixer - 47 - 4.3 Establishment of pyogenic liver abscess model -49 - 4.3.1 Cell culture - 49 - 4.3.2 Cell preparation for DEP manipulation - 50 - 4.3.3 Preparation of bacterial suspension for HepG2 infection - 50 - 4.3.4 Adhesion and cell viability tests - 51 - 4.3.5 Surface pretreatment - 51 - 4.3.6 Instrument setup - 51 - 4.3.7 The process of pyogenic liver abscess model -53 - 4.4 Drug testing for pyogenic liver abscess - 55 - 4.4.1 Preparation of antioxidant - 55 - 4.4.2 Drug testing - 55 - Chapter 5- Results - 57 - 5.1 The results of gradient experiments - 57 - 5.2 The results of acoustic micromixer - 59 - 5.3 The results of on-chip pyogenic liver abscess model -60- 5.3.1 The results of adhesion and cell viability test-60 - 5.3.2 The parameter setup for DEP operation - 61 - 5.3.3 On-chip demonstration of HepG2 and Klebsiella pneumoniae - 62 - 5.3.4 On-chip pyogenic liver abscess model - 63 - 5.4 The results of drug testing for pyogenic liver abscess model - 65 - Chapter 6 - Conclusions - 66 - Reference - 68 -

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