An in vitro cell invasion assay: Determination of cell surface proteolytic activity that degrades extracellular matrix
An in vitro cell invasion assay: Determination of cell surface proteolytic activity that degrades extracellular matrix
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DOI：
10.1007/bf01540646
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发表时间：
1994-01-01
期刊：
影响因子：
--
通讯作者：
Nakahara, Hirokazu
中科院分区：
文献类型：
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作者：
Chen, Wen-Tien;Yeh, Yunyun;Nakahara, Hirokazu
描述了一种便于分析肿瘤细胞体外侵袭表型的相关形态和生化方法。该方法包括标记的基质底物来测量细胞表面蛋白的活性，并使用荧光标记的纤维连接蛋白(或其他纯化的细胞外基质成分)的共价连接到交联的明胶底物的表面。我们基于两个标准研究了组织培养细胞的侵袭性：细胞形成侵袭通道并在交联明胶薄膜上形成表面凹陷的能力，可以通过差示干涉对比显微镜和透射电子显微镜观察到；以及它们通过荧光显微镜局部降解覆盖在松散交联性明胶薄膜上的纤维连接蛋白或其他基质成分的能力。在使用这种方法时，我们已经证明了细胞表面蛋白水解酶的存在，它可能在细胞侵入细胞外基质中起重要作用。此外，我们还鉴定了多种细胞类型，包括由Rous肉瘤病毒转化的胚胎成纤维细胞，恶性人类黑色素瘤细胞系，乳腺癌细胞系，神经小胶质细胞，以及培养中侵袭的神经脊细胞。
A correlative morphological and biochemical method is described that facilitates assaying the invasive phenotype of tumor cells in vitro. The method involves a labeled matrix substrata to measure the activity of cell surface proteases, and uses the covalent linkage of fluorescence-labeled fibronectin (or other purified extracellular matrix components) to the surface of crosslinked gelatin substrata. We have investigated the invasiveness of tissue culture cells based on two criteria: the ability of the cells to form invadopodia and create surface indentations into crosslinked gelatin films that can be visualized by differential interference contrast microscopy and transmission electron microscopy, as well as their ability to locally degrade fibronectin or other matrix components coated on loosely crosslinked gelatin films by fluorescence microscopy. In using this method, we have demonstrated the existence of cell surface proteases that may be important in the invasion of cells into the extracellular matrix. Also, we have identified various cell types including embryonic fibroblasts transformed by Rous sarcoma virus, malignant human melanoma cell lines, breast carcinoma cell lines, neural microglia, and neural crest cells that are invasive in culture.