A genetically encoded bioluminescent indicator for illuminating proinflammatory cytokines.
A genetically encoded bioluminescent indicator for illuminating proinflammatory cytokines.
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DOI：
10.1016/j.mex.2016.06.001
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发表时间：
2016
期刊：
影响因子：
1.9
通讯作者：
Umezawa Y
中科院分区：
文献类型：
--
作者：
Kim SB;Ozawa T;Umezawa Y
我们介绍了一种基于NF-κB核转运的细胞因子活性评价方法。制备了一对生物发光指示剂，用于赋予宫颈癌来源的HeLa细胞的细胞因子敏感性。该原理是基于通过与来自集胞藻属PCC 6803的DnaE内含肽的蛋白质剪接来重建肾形海肾荧光素酶（RLuc）的分裂片段。将HeLa细胞中如此重构的RLuc的生物发光强度用作细胞因子活性的量度。利用本方法，我们评估了基于NF-κB在携带指示物的人宫颈癌源性HeLa细胞中的核转运的各种细胞因子的活性。本研究建立的评价细胞因子活性的方法对药物活性的监测和指导NF-κB相关疾病的治疗具有潜在的临床应用价值。该方法突出了我们的原始出版物，分析的实验程序。2006，359，147-149和Proc.Natl.Biochem.2006，359，147-149。Acad. Sci.联合S. A. 2004，101，11542.该方法的总结是：在刺激后2小时内测定细胞因子活性。暂时失活的分裂荧光素酶片段通过蛋白质剪接重建。NF-κB的核运输被照亮以测量配体驱动的活性。
We introduce a method to evaluate the activities of cytokines based on the nuclear transport of NF-κB. A pair of bioluminescent indicators was made for conferring cytokine sensitivity to cervical carcinoma-derived HeLa cells. The principle is based on reconstitution of split fragments of Renilla reniformis luciferase (RLuc) by protein splicing with a DnaE intein from Synechocystis sp. PCC6803. The bioluminescence intensity of thus reconstituted RLuc in the HeLa cells was used as a measure of the activities for cytokines. With the present method, we evaluated the activities of various cytokines based on the nuclear transport of NF-κB in human cervical carcinoma-derived HeLa cells carrying the indicators. The present approach to evaluating the activities of cytokines may provide a potential clinical value in monitoring drug activity and directing treatment for various diseases related with NF-κB. The method highlights the experimental procedure from our original publications, Anal. Biochem. 2006, 359, 147–149 and Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11542. The summary of the method is: Cytokine activities are determined within 2 h after stimulation. Temporarily inactivated split-luciferase fragments are reconstituted by protein splicing. Nucleartrafficking of NF-κB was illuminated for gauging the ligand-driven activity.