Study of Japanese flounder cytokine gene promoter functions

肿瘤坏死因子被认为是免疫调节和炎症反应的主要介质。然而，对肿瘤坏死因子介导的网络或鱼类中的信号级联知之甚少。本研究将含有2.4kb肿瘤坏死因子(TNF)启动子和绿色荧光蛋白(GFP)基因启动子区域的重组表达载体导入牙鲆肾Yo-K细胞、HINAE细胞和斑马鱼受精的Yo-K细胞中，研究其在细胞系和斑马鱼胚胎中的基因表达调控。YO-K细胞和HINAE细胞与脂多糖共同孵育24 h后，两种细胞表达绿色荧光蛋白的细胞数和绿色荧光蛋白的量均增加。脂多糖可诱导Yo-K细胞和HINAE细胞GFP基因转录。在Yo-K细胞中，GFP的表达水平从刺激后3h到6h逐渐下降，而在HINAE细胞中的表达水平则相反。当转基因斑马鱼胚胎暴露于脂多糖时，绿色荧光蛋白在整个斑马鱼胚胎表面都有表达，Western印迹分析还表明，在内毒素浓度为140μg/ml时，斑马鱼胚胎中绿色荧光蛋白的表达水平高于内毒素浓度为0和70μg/ml时的水平，这一结果表明斑马鱼胚胎中转录可能受内毒素识别系统的调控。

Tumor necrosis factor (TNF) is known as a primary mediator of immune regulation and inflammatory response. However, little is known about TNF-mediated networks or signal cascades in fish. In this study, we characterized the regulation of gene expression of Japanese flounder TNF in cell lines and zebrafish embryo.A recombinant plasmid containing the 2.4 kb promoter region of Japanese flounder tumor necrosis factor (TNF) and green fluorescence protein (GFP) was introduced into YO-K cells derived from Japanese flounder kidney, HINAE cells derived Japanese flounder embryos and zebrafish fertilized. YO-K cells and HINAE cells were incubated with lipopolysaccharide (LPS) at 20℃ for 24 h. The number of cells expressing GFP, as well as the amount of GFP protein was increased by LPS stimulation in both cell lines. GFP mRNA transcription was also induced by LPS stimulation in both YO-K cells and HINAE cells. In YO-K cells, expression level of GFP decreased gradually from 3 h to 6 h post stimulation, while a reverse trend was observed in HINAE cells. When the transgenic zebrafish embryos were exposed to LPS, GFP was expressed in the whole zebrafish embryonic surface and Western blot analysis also showed that the level of the expressed GFP protein in zebrafish embryo was higher at a LPS concentration of 140 μg/ml than at LPS concentrations of 0 and 70 μg/ml. These results indicate that transcription regulated by the Japanese flounder TNF promoter could be under the control of the LPS-recognition system in zebrafish embryos.