QUANTITATION OF CYTOKINE-STIMULATED MIGRATION OF ENDOTHELIUM AND EPITHELIUM BY A NEW ASSAY USING MICROCARRIER BEADS
QUANTITATION OF CYTOKINE-STIMULATED MIGRATION OF ENDOTHELIUM AND EPITHELIUM BY A NEW ASSAY USING MICROCARRIER BEADS
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DOI：
10.1016/0014-4827(90)90205-o
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发表时间：
1990-01-01
影响因子：
3.7
通讯作者：
GOLDBERG, ID
中科院分区：
文献类型：
--
作者：
ROSEN, EM;MEROMSKY, L;GOLDBERG, ID
最近的研究已经确定了一组细胞因子，它们似乎是非白细胞哺乳动物细胞中细胞移动性的细胞特异性调节剂。一个例子是分散因子 (SF)，这是一种由培养的成纤维细胞和血管平滑肌细胞产生的可溶性蛋白质，它会导致紧密、粘性的上皮细胞集落的扩散和分离（“分散”）。 SF 作用的研究受到限制，因为散射程度难以定量，并且散射测定不能用于研究未形成紧密、粘性集落的潜在靶细胞。我们开发了一种简单、定量的 SF 刺激迁移率测定方法，其基础是靶细胞从微载体珠上迁移到 24 孔板的塑料培养表面上。我们发现，源自ras转化3T3细胞的粗制和部分纯化的SF刺激上皮细胞和血管内皮细胞的迁移，但不刺激生产者或非生产者成纤维细胞的迁移。在阳离子交换色谱上共纯化的分散和迁移刺激活性；并且无论 SF 纯度如何，刺激程度与散射滴度密切相关。内皮细胞从珠子的迁移虽然对 SF 极其敏感，但不受血清浓度（1% 至 10%）、各种纯化生长因子或纤连蛋白的影响。在测定孵育期间，珠子的散射和迁移均被放线菌酮(0.1μg/ml)阻断，表明这些过程需要蛋白质合成。微载体珠测定可能是研究 SF 刺激细胞迁移的生化机制的有用定量工具。
Recent studies have identified a group of cytokines which appear to be cell-specific regulators of mobility in nonleukocytic mammalian cells. One example is scatter factor (SF), a soluble protein(s) produced by cultured fibroblasts and vascular smooth muscle cells which causes spreading and separation ("scattering") of tight, cohesive colonies of epithelial cells. Studies of SF action have been limited because the degree of scattering is difficult to quantitate and because scattering assays cannot be used to study potential target cells that do not form tight, cohesive colonies. We developed a simple, quantitative assay of SF-stimulated mobility based on migration of target cells off microcarrier beads onto plastic culture surfaces in 24-well plates. We showed that crude and partially purified SF derived from ras-transformed 3T3 cells stimulates migration of both epithelial and vascular endothelial cells but not of producer or nonproducer fibroblasts. Scatter and migration-stimulating activities copurified on cation exchange chromatography; and the degree of stimulation was closely correlated with scattering titer regardless of SF purity. Migration of endothelial cells from beads, while extremely sensitive to SF, was not affected by serum concentrations (1 to 10%), various purified growth factors, or fibronectin. Both scattering and migration from beads were blocked by cycloheximide (0.1 .mu.g/ml) during assay incubation, suggesting that these processes require protein synthesis. The microcarrier bead assay may be a useful quantitative tool to study the biochemical mechanisms of SF-stimulated cell migration.