細胞位於生物體內時是被一複雜的聚合物所包覆即所謂的細胞外間質。膠原蛋白是構成血管壁的細胞間質中最主要的結構蛋白。在平滑肌細胞分化過程中，平滑肌細胞會藉由表現許多分化的指標蛋白例如smooth muscle α-actin (SM-α-actin)、 SM22、 calponin、 h-caldesmon (h-CaD) 和 smooth muscle myosin heavy chain (SM-MHC)從合成型表現型轉變成收縮型表現型.。這個研究主要探討第一型膠原蛋白的聚合狀態和軟硬度對於培養的平滑肌細胞行為的作用。老鼠血管平滑肌細胞培養在有覆蓋單體的膠原蛋白 (mC) 或覆蓋聚合體的膠原蛋白 (pC) 或聚合體的膠原蛋白凝膠 (pG) 的培養皿上作為實驗組，培養在一般培養皿 (uC) 當成控制組。本實驗中檢視了肌動蛋白纖維 (actin filament)、微管蛋白 (microtubule) 和 SM-α-actin在細胞中的分布。實驗結果顯示，血管平滑肌細胞伸展的比較大當培養在單體的膠原蛋白上時，而培養在聚合體的膠原蛋白上時細胞則變的比較小且有許多絲狀偽足 (filopodia)。Phallodin染色結果顯示肌動蛋白纖維的分布在細胞培養在uC和mC上時，主要是以應激纖維 (stress fiber) 的形式呈現。而培養在pG和pC上時，有80%以上細胞中的肌動蛋白纖維會形成網狀 (meshwork) 的構型，並且出現許多類似粘著斑 (focal adhesion) 的點狀構造。同樣的結果也可在SM-α-actin的分布中觀察到。而微管蛋白 (microtubules)的分布情形在此4種不同的培養條件下都無顯著的差異。為了探討由第一型膠原蛋白所引發的訊息傳遞路徑，我們檢視了FAK的表現和活化以及粘著斑相關蛋白(focal adhesion-associated protein ) vinculin的在細胞中的分布情形。細胞培養在單體的或聚合體的膠原蛋白上時，FAK皆有磷酸化 (phosphorylation)的情形，但表現量在pG上則是下降的。Vinculin最主要是跟肌動蛋白應激纖維 (stress fiber)而不與網狀的肌動蛋白纖維構型有co-localize。為了了解integrin在血管平滑肌細胞培養在不同形式的膠原蛋白後，其呈現不同肌動纖維蛋白構型所扮演之角色，我們檢視了β1 integrin 在細胞中的分布，以及將β1或α1或α2 integrin抑制後對於肌動蛋白的構型是否有所影響。β1 integrin會形成粘著斑並與肌動蛋白應激纖維和網狀構造皆有co-localize。血管平滑肌細胞攤開在單體的或聚合體的膠原蛋白，以及絲狀偽足和肌動蛋白應激纖維的形成都需要β1 integrin和部分的α1 integrin參與。血管平滑肌細胞分化指標蛋白則利用西方點墨法偵測其表現量。血管平滑肌細胞培養在聚合體膠原蛋白上時，其分化指標蛋白例如: SM-MHC或h-CaD的表現量則是下降的，然而SM-α-actin的表現量無顯著的差異。由以上實驗結果顯示，不同聚合狀態的第一型膠原蛋白是透過β1 integrin的作用進而影響血管平滑肌細胞的外型，表現型的轉變以及肌動蛋白的構型。

Most cells in multicellular organisms are surrounded by complex structural molecules that make up the extracellular matrix (ECM). Collagens are major structural components of the extracellular matrix of the artery wall. During smooth muscle cells (SMCs) differentiation, immature SMCs develop from synthetic phenotype to contractile phenotype characterized by the expression of various differentiation markers such as smooth muscle -actin (SM-α-actin), SM22, calponin, h-caldesmon (h-CaD) and smooth muscle myosin heavy chain (SM-MHC). SMCs cultured on polymerized collagen retain their contractile phenotype and mimic many of the characteristics of medial SMCs in vivo whereas SMCs cultured on monomeric collagen are proliferative. This study examined the effect of type I collagen polymerization and stiffness on the behavior of cultured SMCs. Rat aortic SMCs (RASMCs) were cultured on monomeric collagen-coated (mC), polymeric collagen-coated (pC) or polymeric collagen gel (pG) with uncoated dish as control. The distribution of actin filament, microtubules, and SM-α-actin was examined. RASMCs appeared more extended on mC but smaller and with many filopodia on polymeric collagen. Actin filaments detected by phalloidin mainly appeared as stress fibers traversing the cell in RASMCs of mC and control. In contrast, actin filaments formed a meshwork in more than 80% of RASMCs cultured on polymeric collagen, exhibiting prominent structures analogous to focal adhesions. Similar results were observed in SM-α-actin distribution. No difference was detected in microtubule distribution among different conditions. To investigate the signaling pathways from type I collagen, FAK phosphorylation and expression and the distribution of focal adhesion-associated protein, vinculin, were examined. FAK was phosphorylated in RASMCs cultured on both monomeric and polymeric collagen, but the expression of FAK was decreased in RASMCs cultured on pG. Vinculin was co-localized with actin filament along stress fibers but not at actin filament meshwork. To examine the role of integrin in actin filament organization in RASMCs cultured on different forms of collagen, the actin filament organization was examined by phalloidin staining following functional blocking of β1, α1 or α2 integrin with specific antibodies. Using immunofluorescence, β1 integrin was detected at focal adhesions, exhibiting partial co-localization with actin stress fibers and actin meshwork. Functional blocking of β1, α1 or α2 integrin with specific antibody showed that β1 integrin and at least in part, α1 integrin were required for RASMCs spreading, filopodia and actin stress fiber formation in RASMCs cultured on different forms of collagen. The expression of VSMC differentiation markers was examined by Western blot. The expression of differentiation markers, SM-MHC and h-CaD, but not SM-α-actin, decreased in RASMCs cultured on polymeric collagen. These results suggest that the polymerization state of type I collagen substratum modulates cell morphology, phenotypic change and actin filament organization of VSMC in β1 integrin-dependent manner.

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