(18.207.134.98) 您好!臺灣時間:2019/10/23 23:49
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
本論文永久網址: 
line
研究生:張凱婷
研究生(外文):Kai-Ting Chang
論文名稱:CharacterizationsofHepaticstellatecellstreatedwithTaiwanofunguscamphoratamycelia
論文名稱(外文):Characterizations of Hepatic stellate cells treated with Taiwanofungus camphorata mycelia
指導教授:翁慶豐翁慶豐引用關係
指導教授(外文):Ching-Feng Weng
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物學類
畢業學年度:96
語文別:英文
論文頁數:88
外文關鍵詞:Hepatic stellate cellsFE-SEMTM-AFMT. camphorata2-D gel
相關次數:
  • 被引用被引用:0
  • 點閱點閱:599
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
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
Akamatsu S, Watanabe A, Tamesada M, Nakamura R, Hayashi S, Kodama D, Kawase M, Yagi K 2004. Hepatoprotective effect of extracts from Lentinus edodes mycelia on dimethylnitrosamine-induced liver injury. Biological & Pharmaceutical Bulletin 27(12): 1957-1960.
Burridge K, Wennerberg K 2004. Rho and Rac take center stage. Cell 116(2): 167-79.
Canbay A, Higuchi H, Bronk S F, Taniai M, Sebo T J, Gores G J 2002. Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and fibrosis. Gastroenterology 123(4): 1323-30.
Cassiman D, van Pelt J, De Vos R, Van Lommel F, Desmet V, Yap S H, Roskams T 1999. Synaptophysin: A novel marker for human and rat hepatic stellate cells. Am J Pathol 155(6): 1831-9.
Chen C H, Yang S W, Shen Y C 1995. New steroid acids from Antrodia cinnamomea, a fungal parasite of Cinnamomum micranthum. J Nat Prod 58(11): 1655-61.
Chen J J, Lin W J, Liao C H, Shieh P C 2007. Anti-inflammatory benzenoids from Antrodia camphorata. J Nat Prod 70(6): 989-92.
Chiang H C W, D. P.; Cheng, I. H.; Ueng, C. H. 1995. A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia cinnamommea. . Phytochemistry 39: 613-616.
Elsharkawy A M, Oakley F, Mann D A 2005. The role and regulation of hepatic stellate cell apoptosis in reversal of liver fibrosis. Apoptosis 10(5): 927-39.
Fischer R, Cariers A, Reinehr R, Haussinger D 2002. Caspase 9-dependent killing of hepatic stellate cells by activated Kupffer cells. Gastroenterology 123(3): 845-61.
Friedman S L, Roll F J, Boyles J, Bissell D M 1985. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci U S A 82(24): 8681-5.
Greenwel P, Rubin J, Schwartz M, Hertzberg E L, Rojkind M 1993. Liver fat-storing cell clones obtained from a CCl4-cirrhotic rat are heterogeneous with regard to proliferation, expression of extracellular matrix components, interleukin-6, and connexin 43. Lab Invest 69(2): 210-6.
Gressner A M, Weiskirchen R 2006. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med 10(1): 76-99.
Hall A 1998. Rho GTPases and the actin cytoskeleton. Science 279(5350): 509-14.
Hautekeete M L, Geerts A 1997. The hepatic stellate (Ito) cell: its role in human liver disease. Virchows Arch 430(3): 195-207.
Higgins G M, Anderson R M 1931. Experimental pathology of the liver I : restoration of the liver of the white rat following partial surgical removal. Arch Pathol 12: 186–202.
Hill M A, Sun Z, Martinez-Lemus L, Meininger G A 2007. New technologies for dissecting the arteriolar myogenic response. Trends Pharmacol Sci 28(7): 308-15.
Hseu Y C, Chang W C, Hseu Y T, Lee C Y, Yech Y J, Chen P C, Chen J Y, Yang H L 2002. Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes. Life Sci 71(4): 469-82.
Hsiao G, Shen M Y, Lin K H, Lan M H, Wu L Y, Chou D S, Lin C H, Su C H, Sheu J R 2003. Antioxidative and hepatoprotective effects of Antrodia camphorata extract. J Agric Food Chem 51(11): 3302-8.
Ikeda K, Kawada N, Wang Y Q, Kadoya H, Nakatani K, Sato M, Kaneda K 1998. Expression of cellular prion protein in activated hepatic stellate cells. Am J Pathol 153(6): 1695-700.
Imai K, Senoo H 1998. Morphology of sites of adhesion between hepatic stellate cells (Vitamin A-storing cells) and a three-dimensional extracellular matrix. Anatomical Record 250(4): 430-437.
Kawada N, Kristensen D B, Asahina K, Nakatani K, Minamiyama Y, Seki S, Yoshizato K 2001. Characterization of a stellate cell activation-associated protein (STAP) with peroxidase activity found in rat hepatic stellate cells. J Biol Chem 276(27): 25318-23.
Knittel T, Kobold D, Saile B, Grundmann A, Neubauer K, Piscaglia F, Ramadori G 1999. Rat liver myofibroblasts and hepatic stellate cells: different cell populations of the fibroblast lineage with fibrogenic potential. Gastroenterology 117(5): 1205-21.
Knook D L, Seffelaar A M, de Leeuw A M 1982. Fat-storing cells of the rat liver. Their isolation and purification. Exp Cell Res 139(2): 468-71.
Kountouras J, Billing B H, Scheuer P J 1984. Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol 65(3): 305-11.
Kristensen D B, Kawada N, Imamura K, Miyamoto Y, Tateno C, Seki S, Kuroki T, Yoshizato K 2000. Proteome analysis of rat hepatic stellate cells. Hepatology 32(2): 268-77.
Lee I H, Huang R L, Chen C T, Chen H C, Hsu W C, Lu M K 2002. Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS Microbiol Lett 209(1): 63-7.
Lee J S, Kang Decker N, Chatterjee S, Yao J, Friedman S, Shah V 2005. Mechanisms of nitric oxide interplay with Rho GTPase family members in modulation of actin membrane dynamics in pericytes and fibroblasts. Am J Pathol 166(6): 1861-70.
Li X, Benjamin I S, Alexander B 2002. Reproducible production of thioacetamide-induced macronodular cirrhosis in the rat with no mortality. J Hepatol 36(4): 488-93.
Li X N, Huang C T, Wang X H, Leng X S, Du R Y, Chen Y F, Hou X 1990. Changes of blood humoral substances in experimental cirrhosis and their effects on portal hemodynamics. Chin Med J (Engl) 103(12): 970-7.
Liau Y J, Wen L, Shaw J F, Lin C T 2007. A highly stable cambialistic-superoxide dismutase from Antrodia camphorata: expression in yeast and enzyme properties. J Biotechnol 131(1): 84-91.
Liu D Z, Liang H J, Chen C H, Su C H, Lee T H, Huang C T, Hou W C, Lin S Y, Zhong W B, Lin P J, Hung L F, Liang Y C 2007. Comparative anti-inflammatory characterization of wild fruiting body, liquid-state fermentation, and solid-state culture of Taiwanofungus camphoratus in microglia and the mechanism of its action. J Ethnopharmacol 113(1): 45-53.
Liu D Z, Liang H J, Chen C H, Su C H, Lee T H, Huang C T, Hou W C, Lin S Y, Zhong W B, Lin P J, Hung L F, Liang Y C 2007a. Comparative anti-inflammatory characterization of wild fruiting body, liquid-state fermentation, and solid-state culture of Taiwanofungus camphoratus in microglia and the mechanism of its action. J Ethnopharmacol 113(1): 45-53.
Liu D Z, Liang Y C, Lin S Y, Lin Y S, Wu W C, Hou W C, Su C H 2007b. Antihypertensive activities of a solid-state culture of Taiwanofungus camphoratus (Chang-Chih) in spontaneously hypertensive rats. Bioscience Biotechnology and Biochemistry 71(1): 23-30.
Low T Y, Leow C K, Salto-Tellez M, Chung M C 2004. A proteomic analysis of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers. Proteomics 4(12): 3960-74.
Lull C, Wichers H J, Savelkoul H F 2005. Antiinflammatory and immunomodulating properties of fungal metabolites. Mediators Inflamm 2005(2): 63-80.
Maher J J 2001. Interactions between hepatic stellate cells and the immune system. Semin Liver Dis 21(3): 417-26.
Man K, Su M, Ng K T, Lo C M, Zhao Y, Ho J W, Sun C K, Lee T K, Fan S T 2006. Rapamycin attenuates liver graft injury in cirrhotic recipient--the significance of down-regulation of Rho-ROCK-VEGF pathway. Am J Transplant 6(4): 697-704.
Masamune A, Kikuta K, Satoh M, Satoh K, Shimosegawa T 2003. Rho kinase inhibitors block activation of pancreatic stellate cells. Br J Pharmacol 140(7): 1292-302.
Migita K, Maeda Y, Abiru S, Nakamura M, Komori A, Yokoyama T, Takii Y, Mori T, Yatsuhashi H, Eguchi K, Ishibashi H 2006. Immunosuppressant FK506 inhibits matrix metalloproteinase-9 induction in TNF-alpha-stimulated human hepatic stellate cells. Life Sci 78(21): 2510-5.
Nakatani K, Okuyama H, Shimahara Y, Saeki S, Kim D H, Nakajima Y, Seki S, Kawada N, Yoshizato K 2004. Cytoglobin/STAP, its unique localization in splanchnic fibroblast-like cells and function in organ fibrogenesis. Lab Invest 84(1): 91-101.
Niki T, De Bleser P J, Xu G, Van Den Berg K, Wisse E, Geerts A 1996. Comparison of glial fibrillary acidic protein and desmin staining in normal and CCl4-induced fibrotic rat livers. Hepatology 23(6): 1538-45.
Nobes C D, Hall A 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81(1): 53-62.
Ogawa T, Tateno C, Asahina K, Fujii H, Kawada N, Obara M, Yoshizato K 2007. Identification of vitamin A-free cells in a stellate cell-enriched fraction of normal rat liver as myofibroblasts. Histochem Cell Biol 127(2): 161-74.
Parola M, Robino G, Marra F, Pinzani M, Bellomo G, Leonarduzzi G, Chiarugi P, Camandola S, Poli G, Waeg G, Gentilini P, Dianzani M U 1998. HNE interacts directly with JNK isoforms in human hepatic stellate cells. J Clin Invest 102(11): 1942-50.
Prosser C C, Yen R D, Wu J 2006. Molecular therapy for hepatic injury and fibrosis: where are we? World J Gastroenterol 12(4): 509-15.
Ramadori G, Veit T, Schwogler S, Dienes H P, Knittel T, Rieder H, Meyer zum Buschenfelde K H 1990. Expression of the gene of the alpha-smooth muscle-actin isoform in rat liver and in rat fat-storing (ITO) cells. Virchows Arch B Cell Pathol Incl Mol Pathol 59(6): 349-57.
Reeves H L, Friedman S L 2002. Activation of hepatic stellate cells--a key issue in liver fibrosis. Front Biosci 7: d808-26.
Ridley A J, Hall A 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70(3): 389-99.
Ridley A J, Paterson H F, Johnston C L, Diekmann D, Hall A 1992. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70(3): 401-10.
Schmidt M, Gerlach F, Avivi A, Laufs T, Wystub S, Simpson J C, Nevo E, Saaler-Reinhardt S, Reuss S, Hankeln T, Burmester T 2004. Cytoglobin is a respiratory protein in connective tissue and neurons, which is up-regulated by hypoxia. J Biol Chem 279(9): 8063-9.
She H, Xiong S, Hazra S, Tsukamoto H 2005. Adipogenic transcriptional regulation of hepatic stellate cells. J Biol Chem 280(6): 4959-67.
Shen Y C, Chou C J, Wang Y H, Chen C F, Chou Y C, Lu M K 2004a. Anti-inflammatory activity of the extracts from mycelia of Antrodia camphorata cultured with water-soluble fractions from five different Cinnamomum species. FEMS Microbiol Lett 231(1): 137-43.
Shen Y C, Wang Y H, Chou Y C, Chen C F, Lin L C, Chang T T, Tien J H, Chou C J 2004b. Evaluation of the anti-inflammatory activity of zhankuic acids isolated from the fruiting bodies of Antrodia camphorata. Planta Med 70(4): 310-4.
Song T Y, Yen G C 2002. Antioxidant properties of Antrodia camphorata in submerged culture. J Agric Food Chem 50(11): 3322-7.
Song T Y, Yen G C 2003. Protective effects of fermented filtrate from Antrodia camphorata in submerged culture against CCl4-induced hepatic toxicity in rats. J Agric Food Chem 51(6): 1571-7.
Sun K, Wang Q, Huang X H 2006. PPAR gamma inhibits growth of rat hepatic stellate cells and TGF beta-induced connective tissue growth factor expression. Acta Pharmacol Sin 27(6): 715-23.
Tateaki Y, Ogawa T, Kawada N, Kohashi T, Arihiro K, Tateno C, Obara M, Yoshizato K 2004. Typing of hepatic nonparenchymal cells using fibulin-2 and cytoglobin/STAP as liver fibrogenesis-related markers. Histochem Cell Biol 122(1): 41-9.
Torok N, Wu J, Zern M, Halsted C, French S, Friedman S, Zhan S S 2005. Phagocytosis of apoptotic bodies by hepatic stellate cells occurs in vivo and is an important mechanism in liver fibrogenesis. Gastroenterology 128(4): A682-A682.
Wake K 2006. Hepatic stellate cells: Three-dimensional structure, localization, heterogeneity and development. Proceedings of the Japan Academy Series B-Physical and Biological Sciences 82(4): 155-164.
Wang W, Liu F, Chen N 2007. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists attenuate the profibrotic response induced by TGF-beta1 in renal interstitial fibroblasts. Mediators Inflamm 2007: 62641.
Weber L W, Boll M, Stampfl A 2003. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol 33(2): 105-36.
Wen L, Huang H M, Juang R H, Lin C T 2007. Biochemical characterization of 1-Cys peroxiredoxin from Antrodia camphorata. Appl Microbiol Biotechnol 73(6): 1314-22.
Wu H, Pan C L, Yao Y C, Chang S S, Li S L, Wu T F 2006. Proteomic analysis of the effect of Antrodia camphorata extract on human lung cancer A549 cell. Proteomics 6(3): 826-35.
Yee H F, Jr. 1998. Rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton. Hepatology 28(3): 843-50.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔