AN ASSAY FOR TRANSFORMING GROWTH-FACTOR-BETA USING CELLS TRANSFECTED WITH A PLASMINOGEN-ACTIVATOR INHIBITOR-1 PROMOTER LUCIFERASE CONSTRUCT
AN ASSAY FOR TRANSFORMING GROWTH-FACTOR-BETA USING CELLS TRANSFECTED WITH A PLASMINOGEN-ACTIVATOR INHIBITOR-1 PROMOTER LUCIFERASE CONSTRUCT
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DOI：
10.1006/abio.1994.1042
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发表时间：
1994-02-01
影响因子：
2.9
通讯作者：
RIFKIN, DB
中科院分区：
文献类型：
--
作者：
ABE, M;HARPEL, JG;RIFKIN, DB
转化生长因子-β（TGF-β）是细胞分化、增殖、迁移和蛋白质表达的有效调节剂。这些特性已被利用来创建用于检测成熟生长因子的各种生物测定法。基于TGF-β诱导纤溶酶原激活物抑制剂-1（派-1）表达的能力，我们建立了一种高灵敏度、特异性、非放射性的TGF-β定量生物测定法。貂肺上皮细胞（MLEC）稳定转染含有截短的派-1启动子融合到萤火虫荧光素酶报告基因的表达构建体。向转染子中添加TGF-β（0.2至>30 pM）导致细胞裂解物中荧光素酶活性的剂量依赖性增加。虽然对TGF-β有反应，但该启动子片段仅受其他已知派-1表达诱导剂的最小影响。当与广泛使用的MLEC测定法相比时，该测定法表现出更高的灵敏度和特异性，允许在复杂的生物溶液中定量TGF-β。
Transforming growth factor-β (TGF-β) is a potent regulator of cellular differentiation, proliferation, migration, and protein expression. These properties have been exploited to create a variety of bioassays for detecting the mature growth factor. In this paper, we describe a highly sensitive and specific, nonradioactive quantitative bioassay for TGF-β based on its ability to induce plasminogen activator inhibitor-1 (PAI-1) expression. Mink lung epithelial cells (MLEC) were stably transfected with an expression construct containing a truncated PAI-1 promoter fused to the firefly luciferase reporter gene. Addition of TGF-β (0.2 to >30 pM) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. Although responsive to TGF-β, this promoter fragment was only minimally influenced by other known inducers of PAI-1 expression. When compared to the widely used MLEC assay, this assay demonstrated greater sensitivity and specificity, allowing quantification of TGF-β in complex biological solutions.