2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647156摘要：心房細胞外膠原纖維的增加，會使得心房細胞的間隙加大，導致電氣訊號傳遞的延滯，因而增加心房顫動發生的機會。因此心房顫動和心房細胞外間質的改變有相當大的關係。目前倍頻式光學顯微鏡(harmonics optical microscopy)，這種對組織無破壞性的觀察技術在生物實驗上的應用逐漸增加，藉由三倍頻諧波可直接觀察心房細胞的型態及在三度空間中排列的改變；而藉由二倍頻諧波可觀察膠原蛋白形變與在三度空間中的排列的改變。我們更可以同時使用三倍頻和二倍頻諧波，來直接觀察膠原蛋白和心房細胞相互之間的排列關係。本團隊與台大光電研究所孫啟光教授、中央大學數據分析方法研究中心黃鍔院士共同合作；可以直接觀察心房細胞與細胞外膠原纖維在三度空間中排列的改變；更可以將這些三度空間中排列的變化以亂度來量化與分析；這些結果也已經發表在國際知名期刊（PlosOne 2010;5:e13917）。近年來利用奈米縮胺酸生物支架（peptide nanofiberscaffold/RADA16 (AcN-RADARADARADARADA-CONH2)）來建立三度空間纖維母細胞黏附與分泌膠原纖維的實驗已被報告，利用電刺激來刺激鼠類心房細胞HL-1 以模擬心房顫動也被提及；惟利用奈米縮胺酸生物支架來建立心房細胞與心臟纖維母細胞共同培養的心房顫動模式未被報告。再者，從心房顫動病患之心房心肌細胞培養的研究顯示，結締組織生長因子(connective tissue growth factor)訊息傳遞與心房顫動致病機轉的有明顯之相關。透過Rac1 的影響，可以活化結締組織生長因子，進而透過N-cadherin 與connexin 43 可以對心房心肌細胞與心房膠原纖維產生再塑形(remodeling) ，進而產生心房顫動。有鑑於倍頻式光學顯微鏡這項技術，可以直接長時期連續性的觀察細胞與細胞外膠原纖維排列，而不必破壞細胞，我們建立假說：倍頻式光學顯微鏡可以用來協助建立三度空間心房顫動的細胞模式；並協助探討結締組織生長因子訊息傳遞路徑對心房顫動之致病機轉。第一年研究，我們計畫將奈米縮胺酸生物支架與心房心肌細胞及膠原母細胞共同培養，以倍頻式光學顯微鏡直接觀察，將記錄並比較無電刺激和有電刺激心房心肌細胞下，膠原蛋白在三度空間中排列的差異；同時以西方點墨法及螢光組織染色法測量這些細胞纖維相關生物標記的表現濃度(如第一型膠原蛋白,第三型膠原蛋白,細胞外黏附蛋白與膠原蛋白代謝產物)，並檢測組織液心房利鈉胜肽(atrial natri-uretic peptide)濃度。藉此，以比較1) 無電刺激和心房顫動模式下，這些纖維化生物標記和心房外間質纖維排列亂度的相關性；2)無電刺激和心房顫動模式下的心房組織中膠原蛋白排列亂度的差異與心房利鈉胜肽之相關。第二年研究，記錄並比較無電刺激和有電刺激心房心肌細胞下，這些結締組織生長因子之訊息傳遞路徑(Rac1、CTGF、N-cadherin 與connexin 43)的表現。以倍頻式光學顯微鏡直接觀察，記錄並比較膠原蛋白在三度空間中排列的差異與這些結締組織生長因子訊息傳遞路徑之相關；同時觀察二度空間與三度空間的心房顫動細胞模式下，結締組織生長因子之訊息傳遞有無不同。第三年研究，以以不同濃度血管張力素(angiotensin II)加以刺激這些細胞，並以不同濃度史達汀(statin)類藥品投予；並觀察上述細胞培養模式的變化。以倍頻式光學顯微鏡直接觀察，將記錄並比較無電刺激和有電刺激心房心肌細胞下，膠原蛋白在三度空間中排列的差異；同時測量這些細胞Rac1、CTGF、N-cadherin 與connexin 43 的表現。藉此，以比較不同濃度血管張力素與史達汀對結締組織生長因子之訊息傳遞造成之影響；並進而觀察不同濃度血管張力素與史達汀對三度空間奈米縮胺酸生物支架建構的心房顫動細胞模式之影響。<br> Abstract: Atrial fibrillation is associated with fibrosis and atrial remodeling. Enhancedexpression of extracellular matrix (ECM) proteins in atrium will cause atrialcells separation by fibrotic depositions, which may increase the incidence ofatrial fibrillation. Harmonics-based optical microscopy has been widely appliedin biomedical researches. Third-harmonic-generation can provide morphologicinformation including the distribution of atrial cells and second harmonicgeneration can provide distribution of collagen fibers. By utilizingsecond-harmonic-generation and third-harmonic-generation, we can observecontinuously three-dimensional cell-culture. We have published these previousresults in international famous journal (Plos One 2010; 5: e13917). Recently,using self-assembling nanofiber peptide scaffold for three dimensional cultureof fibroblast has been reported. Cell model for atrial fibrillation has beeninduced by rapid field electrical stimulation in atrial HL-1 cells. However,there is no report about using nanofiber peptide scaffold for 3-dimensionalco-culture model of atrial fibrillation. On the other hand, Rac1-inducedconnective tissue growth factor was reported to regulate connexin 43 andN-cadherin expression in atrial fibrillation. Therefore, we hypothesized thatharmonics-based optical microscopy could be applied for construction of suchmodel and assist the evaluation of the role of Rac1-induced connective tissuegrowth factor in pathogenesis of atrial fibrillation. The study aims of the firstyear are: 1)co-culture the atrial cardiomyocytes (HL-1 cell) and cardiacfibroblasts in the nanofiber peptide scaffold ; 2) to observe the spatialalignment of these cells and collagen fibers with or without rapid field electricalstimulation; 3) to check the serum collagen I, collagen III, fibronectin proteinand collagen type I degradation marker level by quantitative western blottingtechniques and immunohistochemical methods; 4) to assess the concentrationof atrial natri-uretic peptide in culture media. The second year, the signaltransduction pathway of connective tissue growth factor under rapid fieldelectrical stimulation will be checked in 3-dimensional and 2-dimensionalco-culture system. We will also compare the correlation between this signaltransduction pathway and atrial remodeling observed by harmonics-basedoptical microscopy. The third year, under different concentrations of statin andangiotensin, the above studies of the second year will be performed.膠原纖維排列心房顫動倍頻式光學顯微鏡奈米縮胺酸生物支架結 締組織生長因子Collagen fiber alignmentatrial fibrillationharmonics optic microscopynanofiber peptide scaffoldconnective tissue growth factor