用于测量生物活性转化生长因子-β的高度灵敏且特异的生物测定法。
Highly sensitive and specific bioassay for measuring bioactive TGF-beta.
作者信息
Tesseur Ina, Zou Kun, Berber Elisabeth, Zhang Hui, Wyss-Coray Tony
机构信息
Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA.
出版信息
BMC Cell Biol. 2006 Mar 20;7:15. doi: 10.1186/1471-2121-7-15.
BACKGROUND
Transforming Growth Factor-beta (TGF-beta) regulates key biological processes during development and in adult tissues and has been implicated in many diseases. To study the biological functions of TGF-beta, sensitive, specific, and convenient bioassays are necessary. Here we describe a new cell-based bioassay that fulfills these requirements.
RESULTS
Embryonic fibroblasts from Tgfb1-/- mice were stably transfected with a reporter plasmid consisting of TGF-beta responsive Smad-binding elements coupled to a secreted alkaline phosphatase reporter gene (SBE-SEAP). Clone MFB-F11 showed more than 1000-fold induction after stimulation with 1 ng/ml TGF-beta1, and detected as little as 1 pg/ml TGF-beta1. MFB-F11 cells were highly induced by TGF-beta1, TGF-beta2 and TGF-beta3, but did not show induction with related family members activin, nodal, BMP-2 and BMP-6 or with trophic factors bFGF and BDNF. MFB-F11 cells can detect and quantify TGF-beta in biological samples without prior enrichment of TGF-betas, and can detect biologically activated TGF-beta in a cell co-culture system.
CONCLUSION
MFB-F11 cells can be used to rapidly and specifically measure TGF-beta with high sensitivity.
背景
转化生长因子-β（TGF-β）在发育过程和成年组织中调节关键生物学过程，并与多种疾病有关。为了研究TGF-β的生物学功能，需要灵敏、特异且便捷的生物测定方法。在此，我们描述了一种满足这些要求的基于细胞的新生物测定方法。
结果
用由TGF-β反应性Smad结合元件与分泌性碱性磷酸酶报告基因（SBE-SEAP）偶联而成的报告质粒稳定转染Tgfb1-/-小鼠的胚胎成纤维细胞。克隆MFB-F11在用1 ng/ml TGF-β1刺激后显示出超过1000倍的诱导，并能检测低至1 pg/ml的TGF-β1。MFB-F11细胞对TGF-β1、TGF-β2和TGF-β3高度诱导，但对相关家族成员激活素、节点蛋白、骨形态发生蛋白-2和骨形态发生蛋白-6或对营养因子碱性成纤维细胞生长因子和脑源性神经营养因子无诱导反应。MFB-F11细胞无需事先富集TGF-β就能检测和定量生物样品中的TGF-β，并且能在细胞共培养系统中检测生物活化的TGF-β。
结论
MFB-F11细胞可用于快速、特异且高灵敏度地测量TGF-β。
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