臺灣博碩士論文加值系統

肝臟星狀細胞 (Hepatic stellate cells, HSCs) 是造成纖維化的主要原因。肝臟星狀細胞平常為靜止狀態，此時肝臟星狀細胞具有貯存脂肪與維他命A的能力，雖表現少量的細胞外基質(Extracellular matrix, ECM)，但並不會損害肝臟；一旦肝臟受到傷害時，肝臟星狀細胞會受刺激而被活化，活化的肝臟星狀細胞，除貯存能力喪失外，還會大量表現細胞外基質，以修補受損的肝臟。活化態的肝臟星狀細胞會大量的增生，並轉形成似纖維母細胞的形態 (myofibroblast-like cell)。因此使活化態肝臟星狀細胞恢復靜止態為本研究方向。本實驗將分離來自硫代乙醯氨 (thioacetamide, TAA) 誘導之老鼠與正常老鼠的肝臟星狀細胞 (分別命名為THSC和NHSC) 做為後續的實驗。結果發現，利用場發射式電子掃描顯微鏡 (FE-SEM) 在不鍍金的情況下，即可清楚觀察到肝臟星狀細胞的奈米結構形態；使用非接觸式原子力顯微鏡 (TM-AFM)，可在不傷害樣本的情況下，進一步證實場發射式電子掃描顯微鏡所見的形態。肝臟星狀細胞以固態培養的牛樟芝菌絲體經水萃物處理後，可發現肝臟星狀細胞會再度貯存脂肪，其貯存脂肪之基因 PPAR

Hepatic stellate cells (HSCs) play a major role of liver fibrosis. In normal liver HSCs remain in quiescent state, quiescent HSCs (qHSCs) have capability to store fatty acid and Vitamin A (retinoid), and secrete less extracellular matrix (ECM); at the time of liver injury, qHSCs are transdifferentiated into activated HSCs (aHSCs), also known as myofibroblast-like cell. aHSCs lose the Vitamin A and fatty acid storage capability and overproduce ECM during activation and proliferation. The overproduced ECMs were stored in hepatic nodal and space between the cells thus causes liver fibrosis. In this study, HSC was isolated from TAA-induced and non-induced liver. TAA-induced HSC (THSC) and non-induced HSC (NHSC) were observed for their morphology and treating them with solid-state of Taiwanofungus camphorata (T.C.) mycelia water extract. Morphology of NHSC and THSC were observed by staining F-actin and using FE-SEM and TM-AFM technique. Both NHSC and THSC had lamellipodia and filopodia, whereas micro-projections were not seen in both cells. On the contrast, THSC were found to have membrane ruffle and absent in NHSC. NHSC and THSC treated with T.C. mycelia water extract, they restored the function of fatty acid storage, thus PPAR

中文摘要 ……………………………………………………… I
Abstract ………………………………………………………… III
Index ……………………………………………………………. V
Content of figures ……………………………………………... VIII
1. Introduction …………………………………………………. 1
1.1. Role of HSCs in liver …………………………………... 1
1.2. Animal model for fibrosis and cirrhosis ……………… 2
1.3. HSCs type and expression ……………………………... 3
1.4. Microscopic morphology of HSC ……………………... 5
1.5. Taiwanofungus camphorata (T. camphorata) ………….. 7
2. Rationale and Specific Aims ……………………………….. 9
3. Research design and methods …………………………….... 11
3.1. Preparation of cells …………………………………….. 12
3.2. Fluorescence microscopy ………………………………. 13
3.3. Oil Red O …………………………….…………………. 13
3.4. Sirius Red ………………………………………………. 14
3.5. Immunocytochemistry …………..……………………... 15
3.6. Immunofluorescence Microscopy …............................... 16
3.7. Field emission scanning electron microscopy ………… 17
3.8. Tapping-mode atomic force microscopy .……………... 18
3.9. Preparation of Taiwanofungus camphorata ………….. 19
3.10. MTS assay ...…………………………………………… 20
3.11. Western blot ………………………………………...…. 20
3.12. Sample preparation for 2D Gel Electrophoresis ...….. 21
3.13. 2-Dimensional electrophoresis …...…………………... 22
4. Results ……………………………………………………….. 23
4.1. Established cell lines …………………………………… 23
4.2. Identification of HSCs …………………………………. 23
4.3. Characteristics of NHSC and THSC ………………….. 24
4.4. Microscopic morphology ………………………………. 24
4.5. Type and morphology of HSCs treated Taiwanofungus
camphorata (T. Camphorata; T.C.) ................................ 25
4.6. Proteomic assay of T. camphorata treated HSCs …….. 27
5. Discussion …………………………………………………… 29
5.1. Advantage of NHSC and THSC ………………………. 29
5.2. Morphology of NHSC and THSC …………………….. 30
5.3. HSCs treated T.C. ……………………………………… 32
6. Conclusion …………………………………………………... 35
7. Future study ………………………………………………… 37
8. References …………………………………………………… 39

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