李嘉哲2006-07-262018-07-112006-07-262018-07-112000http://ntur.lib.ntu.edu.tw//handle/246246/23509肝硬化的成因是由於膠質及其它細胞間質 異常沉積，造成正常組織結構破壞，引起 肝功能失償。這些間質蛋白是由肝星狀細 胞所製造。間質異常沉積可以因製造增加 或是代謝減少所引起。間質金屬蛋白酵素 (MMP)可分解細胞間質所有成份，是間質代 謝的主要酵素。間質金屬蛋白酵素可經由 間質蛋白酵組織抑制因子(TIMP)加以調節 。肝星狀細胞已知可製造MMP-1 、MMP-2 、 MMP-3 、TIMP-1 及TIMP-2 。根據以往的研究，在肝硬化時可觀查到MMP-2 、TIMP-1及TIMP-2 的增加。但是我們的研究卻顯示 MMP-1 增加，而無MMP-2 、TIMP-1 及TIMP-2 的變化。肝星狀細胞基因的表現受到周圍 細胞及間質的影響，因此研究膠質，MMPs 及TIMPs 的表現應直接從肝組織中擷取肝星狀細胞加以分析，而非從組織中分離細胞或培養後再分析。本計畫利用雷射捕捉顯微分離法來擷取硬 化肝及非硬化中之肝星狀細胞，淬取其RNA ，反轉錄後作競爭性PCR ，以定量MMP 及 TIMP 的表現。我們試用了冷凍組織及福馬 林固定之組織切片做雷射捕捉顯微分離法 (LCM)。LCM 每次約捕捉5 個細胞。以actin 為引子所作之RTPCR 顯示只有在冷凍組織 切片才能粹取出mRNA 。大約要100 個細胞 的mRNA 才作的出actin 之RTPCR 。我們用 抗平滑肌actin 及抗desmin 抗體來分辨肝 星狀細胞。只有在福馬林固定之切片染得 出星狀細胞。由於其細長的形狀及緊靠在 肝細胞旁，使得單獨分離不可能。因此以 雷射捕捉顯微分離法來分析肝星狀細胞基因的表現並不可行。Liver cirrhosis is characterized by pathol ogical deposit of collagen and other excellular matrix proteins resulting in disrupt ion of normal liver architecture and a compromise in liver function. These matrix proteins are synthesize d by activated hepatic stellate cell s (HSC).The accumulation of matrix proteins may reflect increased matrix synthesis and/or decreased matrix degradation.Matrix metalloproteinases (MMP) are able to degrade all of the components of extracellular matrix and are the major enzymes in matix turnvoer.Specific inhibition of MMPs occurs by interaction with the tissue inhibitors of matrix metalloproteinases (TIMP). The activated hepatic stellate cells (HSC) is known to secrete procollagen,thr ee MMPs and two TIMPs:interstitial collagenase (MMP-1),gelatinase A (MMP-2)and stromelysin (MMP-3), TIMP-1 and TIMP-2.In this project we tried to isolate HSC using laser capture microdissection (LCM)to study the expression of MMPs and TIMPs.We tried both frozen liver ti ssue and formalin-fixed tissue. PixCell Laser Capture Microdissection System captured about 5 cells in each laser excitation.RTPCR with actin primer only succeeded in frozen tissue.The detection limit is about 100 cells.We used anti-smooth muscle actin and anti-desmin antibody to indentify HSC.HSC can only be stained in formalin- fixed tissue.The filamentous- shaped HSC made the capture of pure cell impossible.We concluded that at present time using LCM to isolate HSC for study expression of MMP and TIMP is not feasible.application/pdf23140 bytesapplication/pdfzh-TW國立臺灣大學醫學院內科肝硬化星狀細胞間質金屬蛋白分解酵素Liver cirrhosisStellate cellMatrix metaloproteinasereporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/23509/1/892315B002025.pdf