- 學位論文
空間效應對細胞間質及心肌細胞交互作用之影響
Geometrical effect on interaction of external cellular matrix and cardiomyocytes
摘要
心肌細胞外膠原纖維的增加，會使得心肌細胞的間隙加大，導致電氣訊號傳遞的延滯，因而增加心律不整發生的機會。因此心律不整和心房細胞外間質的改變有相當大的關係。我們以心房顫動病人的心房組織為材料，以倍頻式光學顯微鏡觀察及二維傅立葉轉換方式定量化方式分析心房組織中細胞外間質量與質排列的變化，證明細胞外間質排列整齊度的變化會對心肌組織產生心律不整的影響，且變化程度和疾病嚴重度相關。近年來利用奈米縮胺酸生物支架進行三度空間細胞培養被認為具有生物工程及用於組織修復的潛力。但是在建立合適的三度空間細胞培養時需同時考量生物支架本身的架構方式可能會影響心肌細胞排列，影響細胞基因調控及功能變化。我們假設不規則排列的3D支架可模擬不規則排列的細胞外間質，使培養的心肌細胞產生表現型的變化。我們觀察以不規則或規則排列方式設計的3D奈米縮胺酸生物支架與傳統2D方式培養出的心肌細胞差異，可見不規則排列方式設計的3D奈米縮胺酸生物支架(Puramatrix)中心肌細胞轉為不規則收縮，和傳統培養模式相比細胞多為紡錘型，胞內鈣離子濃度增加1.42倍。進一步分析發現細胞膜上鈣離子通道蛋白mRNA表現並無明顯增加，但肌漿網上sarcoplasmic reticulum calcium transport ATPase, ryanodine 2 receptor各增加2.14及2.33倍，進一步分析胞內鈣瞬變時發現肌漿網流出的鈣離子明顯增加，應是ryanodine 2 receptor功能異常造成，和胞內鈣離子濃度增加相關。同時細胞間connexin 43的表現較多，分布偏向細胞側壁，B-type natraretic peptide分泌增加。雖然發炎因子、纖維化相關蛋白表現並無增多，但心肌蛋白表現也明顯增加。我們改以規則排列的生物支架(GoMatrix)重複實驗並未觀察到類似的情形。最後我們將培養出的心肌細胞和小鼠心臟細胞直接以mRNA微陣列晶片比較，以生物資訊學方式分析，發現此類細胞較接近擴張型或肥厚型心肌病變的心肌細胞表現。藉此實驗我們證明培養環境的空間排列會改變細胞的發育表現；此類問題在將三度空間細胞培養用於組織修復時需列入考量。由於奈米縮胺酸生物支架可模擬心肌細胞外間質，此類細胞培養方式有做為心律不整的細胞培養模式的潛力；同時也可做為藥物測試的細胞模式。
並列摘要
Enhanced expression of extracellular matrix (ECM) proteins will cause cardiomyocytes separation by fibrotic depositions, delay electrical signal transduction between cells, which may increase the incidence of arrhythmia. We observed atrial fibrillation patient’s atrial tissue by harmonic microscopy, analyzed the entropy of collagen by 2D Fourier transform method, and proved the disarray of collagen increased with disease severity. Recently, using self-assembling nanofiber peptide scaffold for three-dimensional (3D) culture environments for cardiac tissue engineering or regeneration therapy have become a promising field. While developing a proper 3D scaffold, altered 3D organization may have an influence on cell development and cause different cell behavior, which should be taken into consideration. We hypothesized that disordered nanopeptide scaffolds can mimic the chaotic spatial disarray related to cardiac fibrosis and have arrhythmogenic effects on cardiomyocytes. Primary mouse cardiomyocytes were cultured in 2D traditional and 3D disarray nanopeptide hydrogel scaffold (PuraMatrix) systems. Cardiomyocytes in 3D scaffolds showed irregular spontaneous contractile activity as compared with 2D culture controls. Calcium fluorimetric imaging revealed that basal intracellular calcium level increased 1.42-fold in cardiomyocytes cultured in the 3D scaffold, in vitro. The mRNA levels of calcium transport related channels on cell membrane were not increased but sarcoplasmic reticulum calcium transport ATPase, ryanodine 2 receptor elevated 2.14-fold and 2.33-fold in 3D compared with 2D. Further analysis of intracellular calcium sparks showed increased calcium efflux from sarcoplasmic reticulum into the cytoplasm, which could be related to malfunction of ryanodine 2 receptor. Connexin 43 increased 2.62-fold and immunofluorescence imaging revealed lateralization of the distribution of connexin 43 in 3D group. The secretion of B-type natriuretic peptide also increased in 3D. Although inflammation and fibrosis factors were not elevated, myofiber protein had higher expression in 3D group. These findings were not found in cardiomyocytes cultured in another anisotropic 3-dimensional collagen-GAG scaffold (GoMatrix). The mRNA expression of cells in the 3D group and mice cardiomyocytes were compared by microarray. Bioinformatics analysis showed these cells had similar change as those in hypertrophy or dilated cardiomyopathy. This study demonstrated that nanopeptide scaffold geometric irregularity can induce arrhythmogenic effect on cardiomyocytes. These limitations should be considered during cardiac tissue engineering. This 3D cardiomyocyte culture model will be useful for the design and improvement of engineered tissues for construction of cell model of cardiac arrhythmia, tissue engineering application, regeneration therapy and drug testing.